CN114672502A - Construction method of temperature-controlled cell-free reaction system, plasmid used by method and application - Google Patents

Abstract

The present invention relates to a plasmid comprising: a gene encoding a temperature sensitive element; an operating unit located downstream of the gene encoding the temperature sensitive element and capable of being regulated by the temperature sensitive element; an expression target protein located downstream of the operating unit of foreign genes. The plasmid of the present invention can regulate the expression of the target protein through the operating unit regulated by the temperature-sensitive element. When the temperature rises to a given temperature, the temperature-sensitive element is inactivated so that the operating unit starts to express the target protein. When the temperature is lower than At a given temperature, the temperature-sensitive element inhibits the manipulation unit from initiating expression of the target protein. The present invention further provides a method for constructing a temperature-controlled cell-free synthesis system through the plasmid, which realizes that the expression of the target protein in the cell-free system is regulated by temperature.

Description

Construction method of temperature-controlled cell-free reaction system, plasmid used in the method and application

technical field

The invention belongs to the field of biological reaction systems, and in particular relates to a construction method of a temperature-controlled cell-free reaction system, plasmids used in the method and applications.

Background technique

Bioprocess control is an important issue in the fields of biomanufacturing and genetic engineering. The spatiotemporal activation and regulation of protein synthesis and site-specific biological functions are of great significance in the fields of biotechnology, medicine, and industrial production. A commonly used control method is to use the chemical effects of molecules to regulate gene expression. However, this method also suffers from some disadvantages, including unstable chemical effects\low spatial precision and other undesirable side effects that chemical inducers may bring. Using physical signals such as light and heat to control biological processes is an ideal approach. Compared with the poor permeability of light signals to the body, temperature, as a unique physical signal, can accurately and effectively control the overall and local temperature. Temperature-regulated expression systems rely on a strong and finely regulated promoter, avoiding the use of specialized mediators, toxic or expensive chemical inducers.

Currently, temperature-regulated expression has been successfully used for the production of many recombinant proteins and peptides. Temperature is a very well-defined signal that can be spatially directed by externally triggered modalities such as focused ultrasound, infrared light, and magnetic particle hyperthermia. An ideal thermal bioswitch that can respond to temperature changes should have a sensitive switching effect to abrupt temperature changes. Heat shock proteins were rapidly identified as the most striking examples of selective and inducible transcriptional regulation known in eukaryotic cells. Lipid-mediated conformational changes at different temperatures lead to different enzymatic catalytic properties of membrane-associated proteins, which endow membrane-associated proteins with temperature-sensing properties. Temperature-sensing RNA molecules, called "RNA thermometers," can be designed to flexibly switch to respond to temperature with high sensitivity. In addition, transcriptional repressors, in the biomedically relevant 32-46°C range, provide on-off control of bacterial gene expression thresholds.

However, the aforementioned intracellular regulatory mechanisms in response to temperature changes face some inherent difficulties. Temperature is an important factor affecting enzyme activity and complex cellular metabolism, and temperature changes may lead to changes in cellular metabolic intensity and metabolic flux, which can adversely affect target pathways. Furthermore, interactions between different synthetic pathways in vivo may limit effective control of protein expression. Therefore, a more efficient approach is being sought to avoid unnecessary metabolic pathways interfering with temperature control pathways.

The cell-free protein synthesis (CFPS) system is a platform for the in vitro synthesis of proteins that should already be produced in living organisms and are difficult to produce. CFPS uses exogenous DNA or mRNA as a template to supplement inorganic salts, reactive substances and energy substrates in the system. Under the action of various enzymes provided by cell extracts, protein synthesis can be carried out without the cellular environment. Compared with cellular systems, CFPS can avoid the interference of intracellular metabolic pathways on target metabolic pathways at different temperatures, and improve the specificity of temperature-regulated pathways. Furthermore, the cell-free system is characterized by its simplicity and time efficiency as an open synthetic platform without membrane constraints. They only require the addition of certain substances to the biosynthetic reaction that can be completed in a few hours, allowing rapid exploration and screening of factors in CFPS that are critical to controlling elements. Furthermore, previous studies have shown that cell-free synthetic systems controlled by physical signals are encapsulated in liposomes to mimic the population communication of living cells, and that such responsive artificial cells have the potential to deliver drugs.

SUMMARY OF THE INVENTION

The present invention establishes a temperature-responsive genetic circuit in a CFPS system, thereby controlling protein synthesis in vitro by temperature (Fig. 1). The thermosensitive variant of bacteriophage λ repressor cI857 (abbreviated as cI in the present invention) is used to construct a temperature-controlled cell-free protein synthesis (hereinafter referred to as tcCFPS) system, complete the reconstruction of plasmids and optimize cell extracts and redox conditions. The present invention also establishes artificial cells that can control protein synthesis by temperature.

Specifically, the following technical solutions are provided:

1. A plasmid comprising:

Genes encoding temperature-sensitive elements;

An operator unit located downstream of the gene encoding the temperature-sensitive element that can be regulated by the temperature-sensitive element;

The exogenous gene that expresses the target protein located downstream of the operator unit.

2. The plasmid according to item 1, wherein the manipulation unit that can be regulated by the temperature-sensitive element means that when the temperature rises to a given temperature, the temperature-sensitive element is inactivated so that the manipulation unit starts to express a target protein; And when the temperature is lower than a given temperature, the temperature-sensitive element inhibits the manipulation unit from initiating expression of the target protein, and the given temperature is preferably 37-40°C.

3. The plasmid according to item 1 or 2, wherein the temperature-sensitive element and the manipulation unit are used in conjunction, and the temperature-sensitive element and the manipulation unit are selected from the group consisting of bacteriophage λ repression cI-pR-pL promoter, Either TlpA protein-TlpA promoter, E. coli repressor TetR thermosensitive variant-tet operator, E. coli repressor LacI(Ts)-lacO site+LacI promoter or E. coli heat shock protein-pHSP promoter Preferably, the temperature sensitive element and the operator unit are selected from the bacteriophage lambda repressor cI-pR-pL promoter.

4. The plasmid according to any one of items 1-3, wherein the initial plasmid for constructing the plasmid is selected from pSB3K3 plasmid, pET series plasmid, pGEX series plasmid, pMAL series plasmid, pQE-1 plasmid, pQE- 60 plasmid, pGS-21a plasmid, pCDFDuet-1 plasmid, pRSFDuet-1 plasmid or any one of pBluescriptII series plasmids, preferably pSB3K3 plasmid.

5. The plasmid according to any one of items 1-4, wherein the target protein is selected from fluorescent proteins, vaccine proteins, antibody proteins, biocatalytic enzymes, membrane proteins, polypeptides, cytokine proteins, hormone proteins or complement proteins any one or two or more of them.

6. The plasmid according to item 5, wherein the fluorescent protein is any one or two or more selected from the group consisting of red fluorescent protein, green fluorescent protein, orange fluorescent protein and yellow fluorescent protein.

7. Use of the plasmid according to any one of items 1 to 6 for synthesizing a target protein in a cell-free reaction system.

8. A liposome drug delivery system comprising:

The plasmid according to any one of items 1-4, wherein the target protein is a drug protein;

Cell-free extracts of prokaryotic or eukaryotic cells; and temperature-sensitive proteins

9. A construction method of a temperature-controlled cell-free reaction system, the method comprising the steps:

-Provide cell extracts;

- mixing the cell extract with a plasmid comprising a gene encoding a temperature-sensitive element and a gene downstream of the gene encoding a temperature-sensitive element capable of being regulated by the temperature-sensitive element to form a temperature-controlled cell-free reaction system The operation unit and the exogenous gene expressing the target protein located downstream of the operation unit;

-Optional: adding energy source substances and amino acid mixtures, inorganic salts, transcription and translation auxiliary substances to the temperature-controlled cell-free reaction system;

- optional: adding oxidizing substances and reducing substances to the temperature-controlled cell-free reaction system;

- expressing the target protein in a reaction system at a given temperature.

10. A construction method of a temperature-controlled cell-free reaction system, the method comprising the steps:

- providing a cell extract mixture of cell extracts comprising temperature-sensitive elements and cell extracts not comprising temperature-sensitive elements;

- the cell extract mixture is mixed with a plasmid to form a temperature-controlled cell-free reaction system, the plasmid comprising a manipulation unit that can be regulated by the temperature-sensitive element and an exogenous protein of interest that is located downstream of the manipulation unit Gene;

-Optional: adding energy source substances and amino acid mixtures, inorganic salts, transcription and translation auxiliary substances to the temperature-controlled cell-free reaction system;

- optional: adding oxidizing substances and reducing substances to the temperature-controlled cell-free reaction system;

- expressing the target protein in a reaction system at a given temperature.

11. The method according to item 10, wherein the volume ratio of the cell extract comprising the temperature sensitive element and the cell extract not comprising the temperature sensitive element is 1:2-2:1.

12. The method according to any one of items 9 to 11, wherein the cell extract is derived from Escherichia coli, archaea, malt cells, yeast cells, rabbit reticulocytes, tobacco cells , cellular extracts of insect cells or Chinese hamster ovary cells.

13. The method according to any one of items 9-12, wherein the temperature-sensitive element and the manipulation unit are used in conjunction, and the temperature-sensitive element and the manipulation unit are selected from the group consisting of bacteriophage lambda repressor cI-pR-pL Promoter, TlpA protein-TlpA promoter, E. coli repressor protein TetR thermosensitive variant-tet operator, E. coli repressor protein LacI(Ts)-lacO site+LacI promoter or E. coli heat shock protein-pHSP promoter In any one, preferably, the temperature sensitive element and the operation unit are selected from the phage λ repressor cI-pR-pL promoter.

14. The method according to any one of items 9 to 13, wherein the energy source substance is selected from the group consisting of sucrose, maltose, glucose, glucose-6-phosphate, fructose-1,6-diphosphate, phosphoglycerate, phosphoric acid One or more of creatine, adenosine triphosphate, acetyl phosphate, glutamate, polyphosphate, and phosphoenolpyruvate.

15. The method according to any one of items 9-14, wherein the amino acid mixture is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline acid, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine, arginine and histidine one or more of them.

16. The method according to any one of items 9 to 15, wherein the oxidizing substance is oxidized glutathione (GSSG) and the reducing substance is reduced glutathione (GSH), and the GSSG and GSH are The molar ratio is 2:1-4:1.

17. The method according to any one of items 9-16, wherein the given temperature is 37-40°C.

Invention effect

The present invention establishes a cell-free expression system for temperature-controlled protein synthesis, and by adding the plasmid with temperature-sensitive elements prepared by the present invention, the protein synthesis can be controlled according to a specific temperature. Through the introduction of the temperature-sensitive element of the present invention, unnecessary metabolic pathways in the temperature-regulated expression process in the prior art are avoided to interfere with the temperature-controlled pathway, so that the temperature-controlled cell-free expression system does not produce other additional effects while at the same time more efficient Good to achieve temperature control.

The present invention uses engineered plasmids as a means of introducing replication templates for temperature sensitive elements. Bringing these genetic elements into the reaction system via engineered plasmids proved to be a cost-effective and efficient method.

The invention further optimizes the conditions of the cell-free expression system, and optimizes the redox characteristics by adding different amounts of oxidizing substances and reducing substances, and can obtain a maximum 3.40-fold temperature-controlled induction expression multiple. By changing the types and proportions of cell extracts in the cell-free expression system, a maximum 4.10-fold temperature-controlled inducible expression fold can be obtained. Through the analysis of reaction kinetics, the regulation of pR-pL operon-promoter by cI protein at different temperatures can be more clearly revealed, providing a theoretical basis for the possible application of therapeutic protein synthesis and delivery in the future.

The invention successfully constructs a liposome with protein expression function activated at a specific temperature, and is based on 1-palmitoyl-2 oleyl-sn-glycerol-3-phosphocholine (POPC) and cholesterol, and adopts an oil-in-oil package. Liposomes were constructed by aqueous emulsion transfer method. In order to ensure sufficient reaction time in artificial cells to obtain a certain amount of fluorescent expression, liposomes were prepared at a reaction temperature of more than 12 h. The results from flow cytometry showed that tcCFPS-encapsulated liposomes responded to temperature control. The feasibility of the artificial cell response model to temperature signals is demonstrated.

The plasmid with temperature-sensitive element constructed by the present invention can also control protein synthesis in cells.

Description of drawings

Fig. 1 is the temperature-controlled cell-free protein synthesis system (tcCFPS) established by the present invention;

Figure 2. The regulation mechanism model of tcCFPS using the temperature-sensitive circuit cI-pR-pL;

Fig. 3 A is the structural diagram of plasmid pcI;

Figure 3B is a structural diagram of plasmid pΔcI;

Figure 4 is a comparison chart of the expression of plasmid pcI at different temperatures in a cell system;

Figure 5 is a comparison chart of the expression of plasmid pcI at different temperatures in a cell-free system;

Figure 6 is a graph showing the comparison of the expression of GFP with different molar ratios of GSSG:GSH at different temperatures in a cell-free system;

Fig. 7 is the ratio of the mean fluorescence sum of GFP at different temperatures when the mixed cell extracts of cI-BS cell extracts of different volume fractions are added to the cell-free system;

Figure 8 is a graph showing the concentration of cI-mRNA in tcCFPS at different temperatures;

Fig. 9 is the concentration curve diagram of GFP-mRNA in tcCFPS at different temperatures;

Figure 10 is an artificial cell model containing tcCFPS;

Figure 11 is a graph showing the comparison of the expression of GFP in liposomes at different temperatures;

Figure 12A is a confocal image of Liss RhodPE fluorescence in liposomes at 30°C;

Figure 12B is a confocal image of Liss RhodPE fluorescence in liposomes at 37°C;

Figure 12C is a confocal image of Liss RhodPE fluorescence and GFP fluorescence in liposomes at 30°C;

Figure 12D is a confocal image of Liss RhodPE fluorescence and GFP fluorescence in liposomes at 37°C.

Detailed ways

It should be noted that certain terms are used in the description and claims to refer to specific components. It should be understood by those skilled in the art that the same component may be referred to by different nouns. The description and the claims do not use the difference in terms as a way to distinguish components, but use the difference in function of the components as a criterion for distinguishing. As referred to throughout the specification and claims, "comprising" or "including" is an open-ended term and should be interpreted as "including but not limited to". Subsequent descriptions in the specification are preferred embodiments for implementing the present invention, however, the descriptions are for the purpose of general principles of the specification and are not intended to limit the scope of the present invention. The scope of protection of the present invention should be determined by the appended claims.

The present invention relates in a first aspect to: plasmids.

In a specific embodiment, a plasmid is provided, which comprises: a gene encoding a temperature-sensitive element; an operating unit located downstream of the gene encoding a temperature-sensitive element that can be regulated by the temperature-sensitive element; The exogenous gene that expresses the target protein downstream of the operator unit.

In the context of this specification, a "plasmid" conforms to the general definition in the field of bioengineering, which is a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast, and actinomycetes that exists in the cytoplasm (but yeast Except that the yeast deposit plasmid exists in the nucleus), it has the ability to autonomously replicate, so that it can maintain a constant copy number in the daughter cells and express the genetic information it carries. It is a closed circular double-stranded DNA molecule. Plasmids are not necessary substances for bacterial growth and reproduction, and can be lost by themselves or eliminated by artificial treatment, such as high temperature, ultraviolet rays, etc. The genetic information carried by the plasmid can endow the host bacteria with certain biological traits, which are beneficial for the bacteria to survive under specific environmental conditions. Like bacterial genomes, plasmids also belong to circular double-stranded DNA (covalently closed circular DNA, cccDNA). The present invention uses engineered plasmids as a means of introducing protein replication templates (along with promoters responsive to temperature-sensitive proteins) and temperature-sensitive proteins. Bringing these genetic elements together via plasmids into the reaction system proved to be a cost-effective and efficient method.

In the context of this specification, "operator unit" refers to a promoter or operon, or a tandem promoter-operon in the field of bioengineering.

In the context of this specification, a "promoter" conforms to the general definition in the field of bioengineering, and is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription, which contains the specific binding and transcription initiation requirements for RNA polymerase Most of the conserved sequences are located upstream of the transcription initiation site of structural genes, and the promoter itself is not transcribed. However, some promoters (such as tRNA promoters) are located downstream of the transcription initiation site, and these DNA sequences can be transcribed. Promoters were originally characterized by mutations that increased or decreased the rate of gene transcription. A promoter is generally located upstream of the transcription start site.

In the context of this specification, "operon" conforms to the general definition in the field of bioengineering, and refers to a group of key nucleotide sequences, including an operator, a common promoter, and one or more Structural genes are used as motifs for the production of messenger RNA (mRNA). Operators usually consist of two or more coding sequences, promoter sequences, operator sequences and other regulatory sequences clustered in series in the genome. Initiation sequences are specific DNA sequences that RNA polymerase binds to and initiates transcription. In the specific regions of the promoter sequences of various prokaryotic genes, there are usually some similar sequences in the -10 and -35 regions upstream of the transcription start point, which are called consensus sequences. The consensus sequence of E. coli and some bacterial promoter sequences is TATAAT, also known as Pribnow Box, in the -10 region, and TTGACA in the -35 region. Any base mutation or variation in these consensus sequences can affect the binding of RNA polymerase to the promoter sequence and the initiation of transcription. Thus, the degree of identity to the consensus sequence determines the amount of transcriptional activity of the promoter sequence. Operator sequences are binding sites for prokaryotic repressor proteins. When the operator sequence binds to the repressor protein, it will hinder the binding of RNA polymerase to the initiation sequence, or prevent the RNA polymerase from moving forward along the DNA, inhibit transcription, and mediate negative regulation. There is also a specific DNA sequence in the prokaryotic operon regulatory sequence that can bind to activator proteins to activate transcription and mediate positive regulation.

In a specific embodiment, the manipulation unit that can be regulated by the temperature-sensitive element means that when the temperature rises to a given temperature, the temperature-sensitive element is inactivated so that the manipulation unit starts to express the target protein; and When the temperature is lower than a given temperature, the temperature-sensitive element inhibits the manipulation unit from initiating expression of the target protein, and the given temperature is preferably 37-40°C, for example, it can be 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, 40°C.

In the present invention, the gene capable of encoding a temperature-sensitive element is used in conjunction with the manipulation unit, for example, when the temperature-sensitive element is phage λ repressor cI, the manipulation unit is the pR-pL promoter.

The phage λ repressor cI refers to cI857 described in the document Tunable thermal bioswitches for in vivo control of microbial therapeutics. In the present invention, it is named cI38, abbreviated as cI, that is, in the present invention, "phage λ repressor cI", " "cI857", "cI38", "cI" express the same meaning, the phage λ repressor cI is a well-known temperature-sensitive variant that acts on the tandem pR-pL operator-promoter, which can effectively regulate the downstream cloning of the promoter gene.

In other embodiments of the present invention, the temperature-sensitive element and the operating unit may also be TlpA protein-TlpA promoter, E. coli repressor TetR temperature-sensitive variant-tet operon, E. coli repressor LacI (Ts )-lacO site+LacI promoter or E. coli heat shock protein-pHSP promoter.

In a specific embodiment, the target protein is selected from any one or two or more of fluorescent proteins, vaccine proteins, antibody proteins, biocatalytic enzymes, membrane proteins, polypeptides, cytokine proteins, hormone proteins or complement proteins, For the "target protein" involved in the present invention, there are no particular limitations on molecular weight, chemical and physical properties.

In the context of this specification, "vaccine protein" conforms to the general definition in the field of biotechnology, which covers both subunit vaccines and polypeptide vaccines. At present, recombinant DNA technology makes it possible to obtain a large number of pure antigen molecules. This is a revolutionary change in technology compared to vaccines prepared from pathogens, making the quality easier to control and the price higher. In terms of effects, some subunit vaccines, such as acellular pertussis, HBsAg, etc., have high immunogenicity at low doses; while other vaccines have lower immunity and require stronger adjuvants than aluminum salts. Peptide vaccines are usually manufactured by chemical synthesis techniques. The advantage is that the composition is simpler and the quality is easier to control. However, as the molecular weight and structural complexity of the immunogen decreases, the immunogenicity also decreases significantly. Therefore, these vaccines generally require special structural designs, special delivery systems or adjuvants.

In the context of this specification, "biocatalytic enzymes" conform to the general definition in the field of biotechnology and may also be referred to simply as "enzymes". The chemical nature of enzymes is protein (as an exception, a few are RNA), so it also has primary, secondary, tertiary, and even quaternary structure. According to the different molecular composition, it can be divided into simple enzymes and conjugated enzymes. The enzyme protein in the conjugated enzyme is the protein part, and the cofactor is the non-protein part. Only when the two are combined into a holoenzyme can it have catalytic activity. Enzymes are a very important class of biocatalysts. Due to the action of enzymes, chemical reactions in living organisms can be carried out efficiently and specifically under extremely mild conditions. Many enzymes are also available as drugs, such as asparaginase.

In the context of this specification, "membrane proteins" are in accordance with the general definition in the field of biotechnology, ie refer to proteins contained in biological membranes. Membrane proteins can be basically divided into three categories: external membrane proteins or peripheral membrane proteins, internal membrane proteins or integral membrane proteins and lipid-anchored proteins. Membrane proteins include glycoproteins, carrier proteins and enzymes. Usually, some sugars are attached to the outside of the membrane protein, and these sugars are equivalent to transmitting signals into the cell through changes in the molecular structure of the sugar itself. The functions of membrane proteins are multifaceted. Membrane proteins act as "carriers" to transport substances in and out of cells. Some membrane proteins are specific receptors for hormones or other chemicals, such as those on thyroid cells that receive thyrotropin from the pituitary gland. There are also various enzymes on the membrane surface, so that specific chemical reactions can be carried out on the membrane, such as the synthesis of phospholipids on the endoplasmic reticulum membrane. The recognition function of cells is also determined by proteins on the membrane surface. These proteins are often surface antigens. Surface antigens can be combined with specific antibodies. For example, there is a protein antigen HLA on the surface of human cells, which is a dimer with many changes. Membrane proteins play a very important role in many life activities of organisms, such as cell proliferation and differentiation, energy conversion, signal transduction and material transport. It is estimated that about 60% of drugs target membrane proteins.

In the context of this specification, "polypeptide" can be used interchangeably with "polypeptide drug", which covers endogenous polypeptides and exogenous polypeptides; the former is endogenous polypeptides inherent in the human body, such as enkephalin, thymosin, pancreas Polypeptides, etc.; the latter are such as snake venom, sialic acid, bee venom, frog venom, scorpion venom, hirudin, spirulina toxin derivatives and fungicidal peptides secreted by flies, etc., which can have antitumor, antiviral, antibacterial and other aspects. active.

In the context of this specification, "antibody" conforms to the general definition in the field of biotechnology, that is, an antibody refers to a protein with a protective effect produced by the body due to stimulation by an antigen. It (immunoglobulins are not just antibodies) is a large Y-shaped protein secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, etc. It is only found in In the blood and other body fluids of vertebrates, and the surface of the cell membrane of B cells.

In the context of this specification, "cytokine" conforms to the general definition in the field of biotechnology, that is, refers to the production of immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells ( Endothelial cells, epidermal cells, fibroblasts, etc.) are stimulated to synthesize and secrete a class of small molecular proteins with a wide range of biological activities. Cytokines generally regulate immune responses by binding to the corresponding receptors to regulate cell growth, differentiation and effects. Cytokines (CKs) are low-molecular-weight soluble proteins produced by immunogens, mitogens or other stimulants induced by a variety of cells, which have the functions of regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, and injury tissue repair and other functions. Specifically, it can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors and the like.

In the context of this specification, "complement" conforms to the general definition in the field of biotechnology, that is, a serum protein present in human and vertebrate serum and tissue fluid, which is thermolabile, has enzymatic activity after activation, and can mediate Immune and inflammatory responses. It can be activated by antigen-antibody complexes or microorganisms, leading to lysis or phagocytosis of pathogenic microorganisms.

In yet another specific embodiment, the fluorescent protein is any one or more than two selected from red fluorescent protein, green fluorescent protein, orange fluorescent protein or yellow fluorescent protein.

In the context of this specification, a "fluorescent protein" corresponds to the general definition in the field of biotechnology, which is a marker molecule frequently used in biological research. The earliest green fluorescent protein (green fluorescent protein, GFP) was discovered by Shimomura et al. in 1962 in a jellyfish named Aequorea victoria, and then the second GFP was isolated in marine corals. Among them, jellyfish GFP is a monomer protein composed of 238 amino acids with a molecular weight of about 27KD. The production of GFP fluorescence is mainly in the presence of oxygen. The amide of glycine at position 67 in the molecule attacks the carboxyl group of serine at position 65 by nucleophilic attack. After the dehydrogenation of the α-tyrosine bond of the 5th carbon atom imidazolyl and the 66th tyrosine, the aromatic group is combined with the imidazolyl group, so that the p-carboxybenzoate oxazolinone chromophore is formed in the GFP molecule and emits fluorescence . After figuring out this principle, GFP has been widely used in biological research. Various manufacturers such as Promega, Stratagene (including the orange protein preparation technology from the Chinese University of Hong Kong), Clontech (now Takara), etc. have produced corresponding products. In particular embodiments of the present invention the green fluorescent protein GFP is used, the sequence of which is listed below.

In a specific embodiment, the present invention constructs pcI plasmid, the partial DNA sequence of which is shown in SEQID NO.7 of the sequence listing, the sequence includes cI gene sequence, pR operon sequence, pL operon sequence and GFP gene sequence in turn.

In another specific embodiment, the present invention constructs the pΔcI plasmid, the partial DNA sequence of which is shown in SEQ ID NO.

The second aspect of the present invention relates to: the application of the above-mentioned plasmid in a cell-free protein synthesis system.

In a specific embodiment, the use of the above plasmid for synthesizing a target protein in a cell-free protein synthesis system is provided, wherein a temperature-sensitive element as a cofactor is also added to the cell-free protein synthesis system.

In a more specific embodiment, the use of the above plasmid for synthesizing a target protein in a cell-free protein synthesis system based on Escherichia coli extract is provided, wherein the cell-free protein synthesis system is further added as a cofactor. The temperature-sensitive element bacteriophage lambda represses cI.

Here, the temperature-sensitive element is introduced by providing a culture containing a plasmid expressing the temperature-sensitive element.

The present invention relates in a third aspect to: various products based on the above-mentioned applications.

In a specific embodiment, a liposome drug delivery system is provided, comprising the above-mentioned plasmid; wherein the target protein is a drug protein; a cell-free extract of prokaryotic or eukaryotic cells; and a temperature-sensitive protein; and the The drug delivery system needs to be stored at low temperature to prevent contamination before administration to the human body.

The present invention relates to a fourth aspect: a method for constructing a temperature-controlled cell-free reaction system.

In a specific embodiment, a method for constructing a temperature-controlled cell-free reaction system is provided, the method comprising the steps of:

-Provide cell extracts;

- mixing the cell extract with a plasmid comprising a gene encoding a temperature-sensitive element and a gene downstream of the gene encoding a temperature-sensitive element capable of being regulated by the temperature-sensitive element to form a temperature-controlled cell-free reaction system The operation unit and the exogenous gene expressing the target protein located downstream of the operation unit;

-Optional: adding energy source substances and amino acid mixtures, inorganic salts, transcription and translation auxiliary substances to the temperature-controlled cell-free reaction system;

- optional: adding oxidizing substances and reducing substances to the temperature-controlled cell-free reaction system;

- expressing the target protein in a reaction system at a given temperature.

The present invention further provides a method for constructing another temperature-controlled cell-free reaction system, which comprises the following steps:

- providing a cell extract mixture of cell extracts comprising temperature-sensitive elements and cell extracts not comprising temperature-sensitive elements;

- the cell extract mixture is mixed with a plasmid to form a temperature-controlled cell-free reaction system, the plasmid comprising a manipulation unit that can be regulated by the temperature-sensitive element and an exogenous protein of interest that is located downstream of the manipulation unit Gene;

-Optional: adding energy source substances and amino acid mixtures, inorganic salts, transcription and translation auxiliary substances to the temperature-controlled cell-free reaction system;

- optional: adding oxidizing substances and reducing substances to the temperature-controlled cell-free reaction system;

- expressing the target protein in a reaction system at a given temperature.

In a specific embodiment, the cell extract containing the temperature sensitive element can be expressed by expressing the plasmid containing the temperature sensitive element to a certain amount in cells, and then according to the method of Wen et al. (A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. Aeruginosa-Infected Respiratory Samples. ACS Synth. Biol. 2017, 6(12), 2293-2301.) prepared extracts of the cells.

In another specific embodiment, the temperature-sensitive element-containing cell extract can also be a temperature-sensitive element directly added to the cell extract that does not contain a temperature-sensitive element.

The cell extract that does not contain a temperature-sensitive element refers to a cell extract in which the plasmid containing the temperature-sensitive element is neither expressed nor added with a temperature-sensitive element, which is also prepared according to the method of Wen et al.

In a preferred embodiment, according to the present invention, the extract of Escherichia coli BL21Star (DE3) expressing cI repressor protein is mixed with the cell extract of Escherichia coli BL21Star (DE3) without cI repressor protein according to different ratios, and The plasmid pΔcI was added to the cell-free system together to form a temperature-controlled cell-free reaction system.

In a specific embodiment, the cI content in the cell extract comprising the temperature sensitive element is 7.2 ug/mL.

In a further preferred embodiment, the volume ratio of the cell extract containing the temperature sensitive element and the cell extract not containing the temperature sensitive element is 1:2-2:1, for example, it can be 1:2, 1:1.5, 1 : 1, 1.5: 1, 2: 1, preferably, the volume fraction of the cell extract containing cI is 0.67, that is, the volume ratio of the cell extract containing the temperature sensitive element but not the cell extract containing the temperature sensitive element 2:1.

In a specific embodiment, the cells refer to Escherichia coli, archaea, malt cells, yeast cells, rabbit reticulocytes, tobacco leaf cells, insect cells or Chinese hamster ovary cells.

In yet another specific embodiment, the cell extract is derived from E. coli, archaea, malt cells, yeast cells, rabbit reticulocytes, tobacco cells, insect cells or Chinese hamster ovary cells cell extracts.

In yet another specific embodiment, the temperature-sensitive element and the manipulation unit are used in combination, and the temperature-sensitive element and the manipulation unit are selected from phage λ repressor cI-pR-pL promoter, TlpA protein-TlpA promoter , Escherichia coli repressor protein TetR temperature-sensitive variant-tet operon, Escherichia coli repressor protein LacI (Ts)-lacO site+LacI promoter or Escherichia coli heat shock protein-pHSP promoter, preferably, described The temperature sensitive element and the operating unit are selected from the phage lambda repressor cI-pR-pL promoter.

In yet another specific embodiment, the energy source substance can be sucrose, maltose, glucose, glucose-6-phosphate, fructose-1,6-diphosphate, phosphoglycerate, phosphocreatine, adenosine triphosphate, acetyl Phosphoric acid, glutamate, polyphosphate and/or phosphoenolpyruvate.

In yet another specific embodiment, the amino acid mixture comprises glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine acid, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, lysine, arginine and histidine. The transcription and translation auxiliary substances are selected from the group consisting of potassium glutamate, ammonium glutamate, potassium oxalate, magnesium glutamate, oxidized glutathione, reduced glutathione, iodoacetamide, putrescine, One or more of spermine, NAD, NADH, ATP, CTP, GTP, UTP, AMP, CMP, GMP, UMP, CoA, tRNA, and folinic acid.

In a specific embodiment of the present invention, oxidizing substances and reducing substances are added to the temperature-controlled cell-free reaction system to make the temperature control effect better.

Preferably, the oxidizing substance is oxidized glutathione (GSSG) and the reducing substance is reduced glutathione (GSH).

Glutathione (GSH), the most abundant intracellular multifunctional antioxidant, has been shown to be a natural scavenger for free radical detoxification and reactive electrophiles. Furthermore, glutathione disulfide (GSSG), an endogenous peptide, is the oxidized form of GSH. The two redox regulators were added to the tcCFPS containing the pcI plasmid in different proportions to obtain different redox conditions.

In a preferred embodiment, the molar ratio of GSSG to GSH is 2:1-4:1, such as 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5: 1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1.

In a specific embodiment, the given temperature is 37-40°C, such as 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, 40°C.

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the present invention are shown in the drawings, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present invention will be more thoroughly understood, and will fully convey the scope of the present invention to those skilled in the art.

Example 1: Plasmid construction

The bacteriophage lambda repressor cI is a well-known temperature-sensitive variant that acts on the tandem pR-pL operator-promoter and can efficiently regulate genes cloned downstream of the promoter. When the temperature is below 37°C (usually 28-32°C), it represses gene expression, while when the host RNA polymerase raises the temperature above 37°C, the mutated repressor is inactivated and transcribed, the regulation The mechanism is shown in Figure 2.

All plasmids used in the present invention were constructed according to standard molecular biology techniques.

In the present invention, the pSB3K3 plasmid (purchased from Addgene (Addgene#78636)37) is used as the backbone, and the green fluorescent protein GFP is selected as the reporter protein (synthesized by GENEWIZ company). The fluorescent reporter gene was cloned by Gibson assembly under the control of the pL/pR promoter, and the expression suppressor cI and the gene containing the temperature control loop of the pR-pL operator promoter and the GFP fluorescent reporter protein were connected to the pSB3K3 plasmid backbone to obtain the plasmid pSB3K3 -cI-GFP (herein referred to as pcI plasmid) as shown in Figure 3A. A plasmid knocking out the cI protein gene was constructed in the same way and named as pSB3K3-GFPΔcI (hereinafter referred to as pΔcI) as a negative control, as shown in Figure 3B.

Plasmid DNA was purified using OMEGA Plasmid Mini Kit and Qiagen Plasmid Maxi Kit. The plasmid sequence was verified by Tianyi Biotechnology.

The promoter, RBS and partial gene sequence information during plasmid preparation are shown in Table 1.

Table 1 Promoter, RBS and part of gene sequence information

See SEQ ID NO. 7 of the sequence listing for the partial DNA sequence of the pcI plasmid, which sequence includes the cI gene sequence, the pR operon sequence, the pL operon sequence and the GFP gene sequence in turn.

The DNA partial sequence of pΔcI plasmid is shown in SEQ ID NO. 8 of the sequence listing, which sequence includes the pR operon sequence, the pL operon sequence and the GFP gene sequence in turn.

Example 2: Expression of plasmids in cell systems

The pcI plasmid and pΔcI plasmid were transformed into E. coli BL21 Star (DE3) and cultured at 30°C and 37°C for 12 hours, respectively. The OD600 and fluorescence intensity of the bacterial solution were then determined. The results are shown in Figure 4. The GFP level of the pcI plasmid at 37°C was higher than that at 30°C, confirming that the constructed pcI plasmid could activate the expression of the target protein at the expected high temperature in cells.

Example 3: Expression of plasmids in cell-free systems

The protein synthesis control ability of tcCFPS was tested by adding the pcI plasmid directly to the cell-free system and initiating the reaction at different temperatures.

- Preparation of Escherichia coli BL21 Star (DE3) cell-free extract (abbreviated as BS)

For overnight culture with E. coli, 200 mL of 2×YT-P medium was inoculated into a 1-L flask at a dilution ratio of 1:20. The culture was added to a 4 l fermenter and incubated at 37°C, 500 rpm for 3.5 hours. Cell beads were washed twice with 100 mL of ice-cold S30A buffer (14 mM mg-glutamic acid, 60 mM k-glutamic acid, 50 mM Tris, pH 7.7) and then centrifuged at 8000 xg for 15 minutes at 4°C. Then, the cell microspheres were resuspended in 30 ml of frozen S30A buffer, transferred to a pre-weighed 50 ml Falcon conical tube, and centrifuged at 10,000 × g for 10 minutes at 4°C. Finally, the tubes were reweighed and snap-frozen in nitrogen prior to storage at -80 °C.

The next day, the frozen cell beads were thawed in ice and then resuspended with 1 ml of S30A buffer per gram of cell beads. The cell suspension was lysed twice by a high-pressure crusher at a pressure of 15,000-20,000 psi, and centrifuged at 12,000 × g for 10 minutes at 4°C to separate the cytoplasm. After centrifugation, carefully transfer the supernatant to a new tube and add 3 μL of 1 M DTT per 1 ml of lysate. Shake for 80 minutes at 37°C, 120 rpm, and endogenous nuclease digests the remaining mRNA. The extract was centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was transferred to a 6-8kDa MWCO dialysis bag, and then in 1L of S30B buffer (14mM Mg-glutamate, 60mM Kglutamate, ~5mM Tris, pH 8.2) at 4°C under dialysis for 3 hours. Centrifuge again at 12,000 x g for 10 minutes at 4°C. The supernatant was collected to obtain a cell-free extract of E. coli BL21 Star (DE3).

- Expression in cell-free systems

The constructed plasmid was added to the cell-free extract, the reporter protein was green fluorescent protein GFP, and the amount of plasmid added was 300 ng, and the cell-free system was subpackaged to form tcCFPS (temperature-controlled cell-free protein synthesis system).

Reactions were initiated at different temperatures. The results are shown in Figure 5. The GFP level of the pcI plasmid at 37°C was higher than 30°C. The constructed pcI plasmid could play the function of high-temperature activation of protein synthesis in the tcCFPS (temperature-controlled cell-free protein synthesis system) of the BS extract.

Example 4: Effects of redox properties of cell-free protein synthesis system on the folding of translated proteins The present invention further verifies the temperature-controlled expression effect of pcI plasmids under different redox conditions.

Glutathione (GSH), the most abundant intracellular multifunctional antioxidant, has been shown to be a natural scavenger for free radical detoxification and reactive electrophiles. Furthermore, glutathione disulfide (GSSG), an endogenous peptide, is the oxidized form of GSH. The two redox regulators were added to the tcCFPS containing the pcI plasmid in different proportions to obtain different redox conditions. Reactions were initiated at different temperatures.

The results are shown in Figure 6. In the reaction system with excessive addition of GSH solution, the GFP expression of the pcI plasmid did not differ significantly at two temperatures of 30 °C and 37 °C, while excessive addition of GSSG solution to tcCFPS, the temperature control of GFP protein expression The effect is better. When the molar ratio of GSSG to GSH is 2, 3, and 4, that is, when the ratio is 2:1-4:1, the temperature control effect of GFP protein expression is more prominent, especially when 3:1, the temperature-controlled induced expression fold is the highest. is 3.40. This is because different GSSG/GSH ratios change the redox potential of the cell-free system and affect the formation of protein disulfide bonds, while partial oxidation (GSSG) conditions can achieve the best temperature control effect in tcCFPS.

Example 5: Effect of temperature control on cell-free systems by expressing the phage lambda repressor cI in cell extracts

In this experiment, the extract containing cI repressor protein was used to test pΔcI. The extract of Escherichia coli BL21Star (DE3) expressing cI (cI content of 7.2ug/mL) repressor protein was compared with the original one without cI repressor protein. The cell extracts were mixed in different proportions and added to the cell-free system together with the plasmid pΔcI. The ratio of GSSG:GSH in the cell-free system was 3:1, and other conditions were the same as those in Example 3. When observing the mixed cell extracts of different volume fractions of cI-BS cell extracts, the expression levels and ratios of the mean fluorescence of GFP at 37 °C and 30 °C, the results are shown in Figure 7, after adding different amounts of cI-BS. In the case of cell extracts, the temperature control effect can be achieved in the cell-free system, indicating that exogenously expressed cI can endow pΔcI with temperature control ability, especially when the volume fraction of the extract containing cI is 0.67. Temperature control effect, the concentration of cI protein at this time is 4.8ug/mL.

Example 6: tcCFPS expression kinetics study

In order to further explore the mechanism of the elevated transcription and protein expression levels of tcCFPS, the tcCFPS system based on Escherichia coli BL21Star (DE3) extract, plasmid as pcI, and GSSG:GSH ratio of 3:1 was used as the observation object. The tcCFPS was explored for 8 hours at different time points, and the expression kinetics of the system was explored combined with the detection of mRNA levels.

- Transcription process of tcCFPS system

Real-time quantitative PCR detection system (qPCR) was used to monitor the transcription process of the above tcCFPS system. The mRNA levels of cI gene transcription (referred to as cI-mRNA here) at different temperatures (30°C, 37°C) were observed respectively. The results are shown in Figure 8. Under the conditions of 30°C and 37°C, the mRNA levels of cI gene transcription In the first 2 hours, it increased sharply, then decreased, and finally reached a stable level. The degradation rate of mRNA and the DNA transcription rate reached a balance. In the stable level stage, the cI-mRNA at 30 °C was significantly higher than that at 37 °C, indicating that the cI gene The amount that can exert inhibitory effect at 37°C is already much lower than 30°C.

At the same time, the GFP gene transcription level (here referred to as GFP-mRNA) was observed, and the results were shown in Figure 9. Although the mRNA level of GFP gene transcription (here referred to as GFP-mRNA) at 37°C was slightly lower than 30°C in the first hour, However, after one hour, GFP-mRNA at 37°C dropped sharply to a lower level and reached equilibrium, indicating that the inhibitory effect of cI protein on the pR-pL operator promoter resulted in lower GFP-mRNA at 30°C, and cI at 37°C. The expression of the protein decreased slowly, indicating that at 37 °C and above, the cI protein was inactivated, and RNA polymerase was transcribed, so that the GFP gene had a higher transcription level.

Example 7: Expression of plasmids in artificial cells

Artificial cells that mimic the structural and functional characteristics of living cells can be used to further study the physical principles of life and promote the development of cell engineering and biomedicine. The development of cell-free systems has greatly promoted the development of artificial cells. The synthesis of proteins and enzymes in artificial cells has been successfully demonstrated. In the present invention, tcCFPS are compartmentalized into artificial cells that can respond to heat signals. This artificial cell with the function of high-temperature initiation protein synthesis can be used as a potential carrier for drug synthesis and delivery, allowing specific sites to synthesize and release therapeutic proteins when abnormal thermal reactions such as fever occur in the body.

- Preparation of lipid solutions

A water-in-oil (w/o) emulsion transfer method was used to create artificial cell chambers containing a cell-free temperature control system. Using a modified version of the protocol presented in previous studies, lipid solutions were used to prepare liposomes.

Dissolve 30 μL POPC chloroform solution (100 mg/mL) and 15 μL cholesterol chloroform solution (100 mg/mL) in 450 μL liquid paraffin, and add 1 μL Liss Rhod (Lissamine alkaline red B sulfonyl chloride) to make lipids red . In order to fully evaporate the chloroform, the solution was heated at 80 °C for 1 h to obtain a lipid solution.

- Preparation of outer and inner solutions containing tcCFPS.

The solution inside consisted of cell-free reagents, 40 mg of glucose was added, prepared in vials, and kept on ice to prevent the reaction from occurring. The outer solution was prepared with the same composition and concentration as the inner solution, no plasmid was added, and the same molar concentration of glucose was added to the outer solution. The concentration of internal and external solutions should be consistent. If the concentration of the external solution is low, it will cause the internal small molecular weight components to leak from the liposome to the outside.

- Preparation of liposomes

100 μL of lipid solution was added to 200 μL of outer solution (LS-OS) and incubated on ice for 30 min to form an oil-water interface. Add 2 μL of inner solution to 150 μL lipid solution (LS-IS), spin for 1 min, and incubate on ice for 20 min. The LS-IS solution was slowly added to the LS-OS solution, centrifuged at 4000g for 15 minutes at 4°C. The liposome suspension in the lower layer was transferred to another 1.5 mL Eppendorf tube, mixed with 200 μL of fresh outer solution, and centrifuged at 4000 g for 10 min at 4°C. Remove the supernatant and suspend the liposome particles in 20-50 μL of the outer solution. The liposomes encapsulating the previously constructed tcCFPS system solution were obtained, as shown in Figure 10.

Example 8

The effect of protein production was examined by flow cytometry (BD LSRFortessa SORP) analysis of the reactions in liposomes encapsulating previously constructed solutions of tcCFPS systems at 30°C and 37°C. In 19 cases of flow cytometry, a single fluorescent signal of GFP was detected. A total of 60,000 data samples were obtained for the measurement. GFP was excited at 488 nm and emission was detected with a 550 ± 30 nm bandpass filter. From the flow cytometry results, as shown in Figure 11, the tcCFPS-encapsulated liposomes responded to temperature control, and the liposomes reacted at 37 °C to produce more fluorescent proteins than those at 30 °C, And the percentage of liposomes in the high fluorescence band is higher.

Example 9

The liposomes reacted as described above were observed by confocal microscope (Zeiss LSM710). Before measurement, vesicles were diluted to appropriate concentrations with dilution buffer (100 mM HEPES-KOH pH 7.6, 24 mM magnesium acetate, 280 mM potassium glutamate, 1.5 mM DTT, 200 mM glucose). A ×63 objective lens was used for microscope observation. The fluorescence images of GFP were obtained through the corresponding filters, and the results showed that the size of the vesicles was usually between 5-20 μm, and some clear fluorescent vesicles were captured at 30°C and 37°C, as shown in Figure 12. The luminescent part in Figures 12A and 12B is the fluorescence emitted by Liss Rhod stained on the liposome membrane. It can be seen from the figure that the liposome morphology is normal at 30°C and 37°C. Figures 12C and 12D It also includes the GFP light-emitting part. The fluorescence emitted by GFP was observed under the excitation wavelength of 488 nm. The results showed that the expression of GFP in the liposome was very high under the condition of 37 °C, and the expression effect of the target protein was very good. Artificial cells expressing intense fluorescence.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments and application fields, and the above-mentioned specific embodiments are only illustrative and instructive, rather than restrictive . Those of ordinary skill in the art can also make many forms under the inspiration of this specification and without departing from the scope of protection of the claims of the present invention, which all belong to the protection of the present invention.

sequence listing

<110> Tsinghua University

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gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc aggaatctgt cgcagacaag 120

atggggatgg ggcagtcagg cgttggtgcc ttatttaatg gcatcaatgc attaaatgct 180

tataacgccg catcgcttac aagaattctc aaagttagcg ttgaagaatt tagcccttca 240

atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc acttagaagt 300

gagtatgagt accctgtttt ttctcatgtt caggcaggga tgctctcacc tgagcttaga 360

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cttgaggacg cacgtcgcct taaagcaatt tatgaaaaaa agaaaaatga acttggctta 180

tcccaggaat ctgtcgcaga caagatgggg atggggcagt caggcgttgg tgccttattt 240

aatggcatca atgcattaaa tgcttataac gccgcatcgc ttacaagaat tctcaaagtt 300

agcgttgaag aatttagccc ttcaatcgcc agagaaatct acgagatgta tgaagcggtt 360

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gggatgctct cacctgagct tagaaccttt accaaaggtg gtgcggaaag gtgggtaagc 480

acaaccaaaa aagccagtga ttctgcattc tggcttgagg ttgaaggtaa ttccatgaca 540

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aagaaactga tcagggatag cggtcaggtg ttttttacaac cactaaaccc acagtaccca 720

atgatcccat gcaatgagag ttgttccgtt gtggggaaag ttatcgctag tcagtggcct 780

gaagagacgt ttggctgaaa atttagctaa acacacatga attttcagat gtgttttatc 840

cgggtactag agccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc 900

gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg 960

gtgggccttt ctgcgtttat atactagaga tgccggccac gatgcgtccg gcgtagagga 1020

tcgagtatca ccgcaaggga taaatatcta acaccgtgcg tgttgactat tttacctctg 1080

gcggtgataa tggttgcatg tactaaggag gttgtatgga acaacgcata accctgaaag 1140

attatgcaat gcgctttggg caaaccaaga cagctaaaag atctctcacc taccaaacaa 1200

tgcccccctg caaaaaataa attcatataa aaaacataca gataaccatc tgcggtgata 1260

aattatctct ggcggtgttg acataaatac cactggcggt gatactgagc acatcagcag 1320

gacgcactga ccaccatgaa ggtgacgctc ttaaaaatta agccctgaag aagggcagca 1380

ttcaaagcag aaggctttgg ggtgtgtgat acgaaacgaa gcattggtta aaaattaagg 1440

agggaattat gcgtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat 1500

tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa 1560

catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc 1620

caacacttgt cactactttc ggttatggtg ttcaatgctt tgcgagatac ccagatcata 1680

tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta 1740

tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata 1800

cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg 1860

gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa 1920

agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac 1980

tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca 2040

accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca 2100

tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca 2160

aataataata ctagagccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 2220

tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct 2280

tcgggtgggc ctttctgcgt ttatatacta gtagcggccg ctgcagtccg gcaaaaaaac 2340

gggcaaggtg tcaccaccct gccctttttc tttaaaaccg aaaagattac ttcgcgttat 2400

gcaggcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2460

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ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 2640

gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 2700

actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 2760

gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct 2820

ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 2880

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atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 3060

gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 3120

ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 3180

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tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata 3360

acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt 3420

ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat 3480

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cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca 3660

acaccttctt cacgaggcag acctcagcgc tagcggagtg tatactggct tactatgttg 3720

gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt 3780

gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg 3840

ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga 3900

agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat 3960

aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac 4020

ccgacaggac tataaagata ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg 4080

ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca 4140

cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc 4200

cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa 4260

agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt 4320

gaagtcatgc gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc 4380

aagccagtta cctcggttca aagagttggt agctcagaga accttcgaaa aaccgccctg 4440

caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac gatctcaaga 4500

agatcatctt attaaggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4560

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 4620

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 4680

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 4740

cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 4800

gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 4860

cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctattc catggtgcca 4920

cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 4980

aggcagaatt tcagataaaa aaaatcctta gctttcgcta aggatgattt ctg 5033

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<223> Description of artificial sequences: artificially synthesized sequences

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gaattcgcgg ccgcttctag agttgactat tttacctctg gcggtgataa tggttgcatg 60

tactaaggag gttgtatgga acaacgcata accctgaaag attatgcaat gcgctttggg 120

caaaccaaga cagctaaaag atctctcacc taccaaacaa tgcccccctg caaaaaataa 180

attcatataa aaaacataca gataaccatc tgcggtgata aattatctct ggcggtgttg 240

acataaatac cactggcggt gatactgagc acatcagcag gacgcactga ccaccatgaa 300

ggtgacgctc ttaaaaatta agccctgaag aagggcagca ttcaaagcag aaggctttgg 360

ggtgtgtgat acgaaacgaa gcattggtta aaaattaagg agggaattat gcgtaaagga 420

gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg 480

cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt 540

aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc 600

ggttatggtg ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc 660

aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg 720

aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag 780

ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac 840

tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac 900

ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa 960

aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa 1020

tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta 1080

acagctgctg ggattacaca tggcatggat gaactataca aataataata ctagagccag 1140

gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 1200

gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt 1260

ttatatacta gtagcggccg ctgcagtccg gcaaaaaaac gggcaaggtg tcaccaccct 1320

gccctttttc tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc tcgctcactg 1380

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 1440

tctcgagtcc cgtcaagtca gcgtaatgct ctgccagtgt tacaaccaat taaccaattc 1500

tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc 1560

aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt 1620

ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca 1680

acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac 1740

gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg 1800

ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga 1860

ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat 1920

cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg 1980

atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc 2040

atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca 2100

gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag 2160

aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc 2220

gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg 2280

cggcctcgag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt 2340

tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca 2400

tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa 2460

ggatcagatc acgcatcttc ccgacaacgc agaccgttcc gtggcaaagc aaaagttcaa 2520

aatcaccaac tggtccacct acaacaaagc tctcatcaac cgtggctccc tcactttctg 2580

gctggatgat ggggcgattc aggcctggta tgagtcagca acaccttctt cacgaggcag 2640

acctcagcgc tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt 2700

gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat 2760

acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc 2820

gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta 2880

acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa 2940

gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata 3000

ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc 3060

ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg 3120

gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg 3180

cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact 3240

ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag 3300

gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca 3360

aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt 3420

cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt attaaggggt 3480

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3540

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3600

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3660

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3720

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3780

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3840

caactttatc cgcctccatc cagtctattc catggtgcca cctgacgtct aagaaaccat 3900

tattatcatg acattaacct ataaaaatag gcgtatcacg aggcagaatt tcagataaaa 3960

aaaatcctta gctttcgcta aggatgattt ctg 3993

Claims (10)

1. A plasmid, comprising:

a gene encoding a temperature sensitive element;

a manipulation unit that is located downstream of the gene encoding a temperature sensitive element and is capable of being regulated by the temperature sensitive element;

a foreign gene expressing a protein of interest located downstream of the manipulation unit.

2. The plasmid according to claim 1, wherein the manipulation unit that can be regulated by the temperature sensitive element means that when the temperature is increased to a given temperature, the temperature sensitive element is inactivated so that the manipulation unit initiates expression of the target protein; and the temperature sensitive element inhibits the manipulation unit from initiating the expression of the target protein when the temperature is lower than a given temperature, preferably 37-40 ℃.

3. The plasmid according to claim 1 or 2, wherein the temperature-sensitive element and the operator are used in combination, and the temperature-sensitive element and the operator are selected from any one of bacteriophage lambda-repressed cI-pR-pL promoter, TlpA protein-TlpA promoter, TetR thermo-sensitive variant-tet operator of E.coli repressor protein, LacI (Ts) -lacO site + LacI promoter of E.coli repressor protein, or E.coli heat shock protein-pHSP promoter, preferably the temperature-sensitive element and the operator are selected from bacteriophage lambda-repressed cI-pR-pL promoter.

4. The plasmid according to any one of claims 1 to 3, wherein the initial plasmid used for constructing the plasmid is selected from any one of the pSB3K3 plasmid, pET series plasmid, pGEX series plasmid, pMAL series plasmid, pQE-1 plasmid, pQE-60 plasmid, pGS-21a plasmid, pCDFDuet-1 plasmid, pRSFDuet-1 plasmid or pBluescriptII series plasmid, preferably pSB3K3 plasmid.

5. The plasmid according to any one of claims 1 to 4, wherein the target protein is selected from any one or more of a fluorescent protein, a vaccine protein, an antibody protein, a biocatalytic enzyme, a membrane protein, a polypeptide, a cytokine protein, a hormone protein, and a complement protein.

6. The plasmid according to claim 5, wherein the fluorescent protein is any one or more selected from the group consisting of a red fluorescent protein, a green fluorescent protein, an orange fluorescent protein and a yellow fluorescent protein.

7. Use of a plasmid according to any one of claims 1-6 in a cell-free reaction system for the synthesis of a protein of interest.

8. A liposomal drug delivery system comprising:

the plasmid of any one of claims 1-4, wherein the protein of interest is a pharmaceutical protein;

Cell-free extracts of prokaryotic or eukaryotic cells; and temperature sensitive proteins.

9. A method for constructing a temperature-controlled cell-free reaction system comprises the following steps:

-providing a cell extract;

-mixing the cell extract with a plasmid comprising a gene encoding a temperature sensitive element and a manipulation unit capable of being regulated by the temperature sensitive element located downstream of the gene encoding the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell-free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature control cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature-controlled cell-free reaction system;

-expressing the protein of interest in a reaction system at a given temperature.

10. A method for constructing a temperature-controlled cell-free reaction system comprises the following steps:

-providing a cell extract mixture of a cell extract comprising a temperature sensitive element and a cell extract not comprising a temperature sensitive element;

-mixing the cell extract mixture with a plasmid comprising a manipulation unit capable of being manipulated by the temperature sensitive element and a foreign gene expressing a protein of interest located downstream of the manipulation unit to form a temperature controlled cell free reaction system;

-optionally: adding an energy source substance, an amino acid mixed solution, inorganic salt and a transcription and translation auxiliary substance into the temperature-controlled cell-free reaction system;

-optionally: adding an oxidizing substance and a reducing substance to the temperature controlled cell-free reaction system;

-expressing the target protein in a reaction system at a given temperature.

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