CN114196699B

CN114196699B - Method for increasing extracellular secretion of plant cell recombinant protein

Info

Publication number: CN114196699B
Application number: CN202111462403.4A
Authority: CN
Inventors: 缪国鹏; 相深; 杨纬经
Original assignee: Liejing Jiaxing Biotechnology Co ltd
Current assignee: Liejing Jiaxing Biotechnology Co ltd
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-12-20
Anticipated expiration: 2041-12-02
Also published as: CN114196699A

Abstract

The invention provides a method for increasing extracellular secretion of a plant cell recombinant protein. The method specifically comprises the following steps: 1) Respectively constructing an expression vector containing an EXO gene and an expression vector containing a gene for coding the recombinant protein, or constructing a co-expression vector containing the EXO gene and the gene for coding the recombinant protein; 2) Transforming a plant cell to secretionally express a recombinant protein; wherein the nucleic acid sequence of the EXO gene is shown as SEQ ID NO.1, and the nucleic acid sequence of the gene for coding the recombinant protein comprises a nucleic acid sequence for coding a secretory signal peptide connected to the N end of the recombinant protein. The invention discovers for the first time that the EXO gene can realize the transportation of recombinant protein positioned in endoplasmic reticulum and extracellular space, and realize the extracellular secretion of the recombinant protein; the EXO gene and the gene for coding the recombinant protein simultaneously transform plant cells, which has a synergistic effect and improves the yield and the secretion rate of the recombinant protein.

Description

Method for increasing extracellular secretion of plant cell recombinant protein

Technical Field

The invention relates to the technical field of DNA recombination, in particular to a method for increasing extracellular secretion of plant cell recombinant protein.

Background

According to statistics, 1/4 of new drugs on the market based on the United states food and drug administration are recombinant protein drugs, and the recombinant protein drugs are up to 151 in 2009. At present, the majority of the clinically used recombinant protein drugs come from mammalian cells and Escherichia coli expression production platforms, and a small part of the drugs come from yeast and insect cells. Various expression systems have advantages and disadvantages, an expression product can be obtained in a short time by using an escherichia coli expression system, the required cost is relatively low, but target protein is often expressed in an inclusion body form, the product is difficult to purify, a prokaryotic expression system is incomplete in post-translational processing and modification system, and the biological activity of the expression product is low; yeast and insect cell expression systems have high protein expression levels and low cost, but the post-translational processing modification system is not identical to that of mammals. The protein produced by the mammalian cell expression system is closer to the natural state, but the expression level is low, and the operation is complicated. Therefore, a new expression system with high expression, economical efficiency, high safety is required for producing these recombinant proteins. Compared with the traditional microbial fermentation and animal cell culture, the method for producing the recombinant protein by using the plant bioreactor has the advantages of wide expression range (5 kD-300 kD), high expression quantity, accurate folding, good uniformity, high biological activity, easy storage, scale and easy production, low production cost, safety, no pollution and the like.

However, if the expression position of the recombinant protein in the plant cell is located intracellularly, many problems are caused. Such as increasing the metabolic burden on the host; or the bioactivity is not good, which causes the effective yield of the recombinant protein to be reduced, and the bioactivity is difficult to ensure. The extracellular secretory expression can better solve the problem. The extracellular secretory expression can improve the bioavailability of the recombinant protein, the extracellular environment is more favorable for the stability and activity of the recombinant protein, the purification is easy, the toxicity to host bacteria and the recombinant protein can be reduced, and the secretory expression can be continued.

However, the current research on increasing extracellular secretion of recombinant proteins focuses mainly on the addition of secretory signal peptides. However, fusion of a secretory signal peptide to a recombinant protein does not effectively enhance the secretion of the recombinant protein, and most of them remain inside the cell (Huang et al, PLOS ONE.2015,10 (10): e 0140812). Therefore, it is an urgent need to develop a method for increasing the extracellular secretion of plant cell recombinant protein, so as to obtain a recombinant protein with high yield, high secretion rate, high purity and low cost.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for increasing the extracellular secretion of a plant cell recombinant protein, which solves the problems of low yield, low secretion rate and difficult purification of products in the production of the recombinant protein in the prior art.

In one aspect of the present invention, there is provided a method for increasing extracellular secretion of a recombinant protein in a plant cell, comprising: the method comprises the following steps:

1) Respectively constructing an expression vector containing an EXO gene and an expression vector containing a gene coding a recombinant protein, or constructing a co-expression vector containing the EXO gene and the gene coding the recombinant protein;

2) Transforming a plant cell to secretionally express a recombinant protein;

wherein, the nucleic acid sequence of the EXO gene is shown as SEQ ID NO.1, and the nucleic acid sequence of the gene for coding the recombinant protein comprises a nucleic acid sequence for coding a secretory signal peptide connected to the N end of the recombinant protein.

The expression vector containing the EXO gene expresses the EXO factor after transforming plant cells; the expression vector containing the gene for coding the recombinant protein expresses the recombinant protein after transforming plant cells; the co-expression vector containing the EXO gene and the gene coding the recombinant protein expresses two proteins of the EXO factor and the recombinant protein after transforming plant cells. The secretory signal peptide connected with the N end of the recombinant protein can guide the synthesized recombinant protein to enter the lumen of the endoplasmic reticulum through the endoplasmic reticulum membrane and is finally secreted to the outside of cells.

Further, the secretory signal peptide comprises NtPR1a-SSP, and the nucleic acid sequence for coding the NtPR1a-SSP is shown as SEQ ID NO. 2.

NtPR1a-SSP recognizes and binds to receptors on the endoplasmic reticulum membrane, thereby directing the recombinant protein into the lumen of the endoplasmic reticulum.

Further, the nucleic acid sequence of the gene encoding the recombinant protein includes a nucleic acid sequence encoding an endoplasmic reticulum retention signal including KDEL or HDEL linked to the C-terminus of the recombinant protein.

KDEL is the Lys-Asp-Glu-Leu amino acid residue sequence and HDEL is the His-Asp-Glu-Leu amino acid residue sequence. When KDEL or HDEL is added to the C-terminal of the recombinant protein, the recombinant protein can be retained in the endoplasmic reticulum.

Further, a non-coding region is connected to the 5 'end of the nucleic acid sequence of the gene for coding the recombinant protein, the non-coding region comprises AtCOR 47' UTR, and the nucleic acid sequence of the gene is shown as SEQ ID NO.3.

The non-coding region, which may also be referred to as a non-translated region, is a regulatory element used to enhance transcription and translation of the recombinant protein.

Furthermore, the EXO gene or the gene encoding the recombinant protein is driven by promoters, and the promoters comprise CaMV35S promoter, enhanced CaMV35S promoter, NOS promoter and ACTIN promoter.

Further, the recombinant protein includes cytokines, antibodies, vaccines, enzymes, blood products.

The method of the invention is suitable for preparing various recombinant proteins by DNA recombination technology, for example, cytokines comprise interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemotactic factors, growth factors and the like; the antibody comprises adalimumab, herceptin antibody, infliximab, etanercept, rituximab, bevacizumab, trastuzumab and the like; the vaccine comprises recombinant influenza virus vaccine, recombinant hepatitis B vaccine, recombinant new coronavirus vaccine and the like; the enzyme includes proteinase K, amylase, protease, glucose oxidase, xylanase, lipase, etc.; the blood products comprise serum albumin, blood coagulation factors and the like, and the recombinant proteins are all suitable for being prepared by the preparation method.

In one embodiment of the present invention, the recombinant protein is interleukin 10, and the nucleic acid sequence of interleukin 10 is SEQ ID No.4.

In another embodiment of the present invention, the recombinant protein is a herceptin antibody, and the nucleic acid sequences of the herceptin antibody are SEQ ID No.5 and SEQ ID No.6.

Further, the plant cells comprise tobacco suspension cells, carrot suspension cells and Arabidopsis suspension cells.

In still another aspect of the present invention, there is provided a vector comprising an expression vector containing an EXO gene and an expression vector containing a gene encoding a recombinant protein; or a co-expression vector containing an EXO gene and a gene encoding a recombinant protein.

In another aspect of the present invention, there is provided a host cell comprising the vector of claim 8.

The recombinant protein can be obtained by secreting the vector into host cells.

In still another aspect of the present invention, there is provided a use of the above-mentioned vector or host cell for the preparation of a recombinant protein drug.

The technical principle of the invention is as follows:

EXO is the extracellular subunit EXO70 family protein E2 of the extracellular positive organelle EXPO (extracellular positive organisms). It was initially discovered that overexpression of Arabidopsis EXO recruits the formation of EXPO, which traps, encapsulates, and transports proteins in the cytoplasm and is secreted extracellularly (e.g., S-adenosylmethionine synthetase 2); however, the other functions of EXPO are not clear. Through further research and exploration, the inventor unexpectedly finds that the EXO and Enhanced Green Fluorescent Protein (EGFP) with different locations construct a co-expression vector, the EXO can not only transport the EGFP which is located and expressed in cytoplasm, but also can transport the EGFP which is located and expressed in endoplasmic reticulum and extracellular; the inventor further finds that EXO overexpression obviously improves the secretion rate and total yield of EGFP protein which is expressed in endoplasmic reticulum and extracellular, and the EXO can promote the secretion of EGFP; for EGFP at different expression positions, the promotion effect of EXO on the localization of EGFP expressed in endoplasmic reticulum and extracellular EGFP is obviously better than that of EGFP expressed in cytoplasm, and the promotion effect of EXO on EGFP expressed in endoplasmic reticulum is most obvious. The inventors speculate that this may be due to the fact that EXO recruits proteins localized in the endoplasmic reticulum when it accumulates in the endoplasmic reticulum intima and adventitia, and that these recruited proteins are transferred into the organelles when the EXO organelles are formed, thereby promoting the extracellular secretion of proteins.

In the subsequent experiment process, KDEL or HDEL is added into the carboxyl terminal of the medicinal recombinant protein, secretory type signal peptide is added into the amino terminal of the medicinal recombinant protein, and the medicinal recombinant protein and EXO are co-expressed, the experiment result is consistent with the EGFP, the extracellular secretion amount and the total yield of the recombinant protein are obviously increased, and the increase range of the secretion amount and the total yield is obviously superior to that of the recombinant protein without the EXO, so that the EXO can promote the secretion of the recombinant protein which is positioned and expressed in the endoplasmic reticulum.

Compared with the prior art, the invention has the following beneficial effects:

1) The invention discovers for the first time that the EXO gene can realize the transportation of recombinant protein positioned in endoplasmic reticulum and extracellular space, and realize the extracellular secretion of the recombinant protein;

2) The EXO gene has obvious promotion effect on the secretion of recombinant proteins positioned in the endoplasmic reticulum and outside cells, and has the most obvious promotion effect on the recombinant proteins positioned in the endoplasmic reticulum;

3) The EXO gene and the gene for coding the recombinant protein simultaneously transform plant cells, which has a synergistic effect, improves the yield and the secretion rate of the recombinant protein, and the effect of the gene is obviously superior to that of the recombinant protein without the EXO gene;

4) The recombinant protein prepared by the method is secreted to the outside of cells, and is easy to extract and purify;

5) The recombinant protein prepared by the method is secreted by plant cells, and can be well detected by an antibody, so that the extracellularly secreted recombinant protein has good integrity and conformation, and therefore, has good activity.

Drawings

FIG. 1 is a diagram of a pYBA1132 expression vector of example 1 of the present invention, in which origin represents an initiation region, nptII is a selection marker gene for encoding neomycin phosphotransferase, T-DNA left border represents a left border sequence of a T-DNA region, NOS promoter represents an NOS promoter, NOS terminator represents an NOS terminator, caMV35S promoter represents a CaMV35S promoter, bamHI and XhoI are cleavage sites, EGFP is a fluorescent marker for encoding enhanced green fluorescent protein, caMV polyA signal represents a terminator, T-DNA right border sequence of a T-DNA region, and REP is a replication origin sequence for encoding REP protein;

FIG. 2 is an EGFP expression vector map in example 1 of the present invention, wherein A is pYBA1132-cytEGFP, B is pYBA1132-sseEGFP, and C is pYBA1132-sseEGFP-KDEL;

FIG. 3 is a map of the EXO and EGFP co-expression vector in example 1 of the present invention;

FIG. 4 is a graph showing the fluorescence effect observed after Agrobacterium-mediated EGFP gene was transformed into tobacco BY-2 cells in example 2 of the present invention, wherein a is callus and b is a cell obtained BY suspension culture of the callus;

FIG. 5 is a graph showing the effect of fluorescence on the expression site of cytoplasmic-localized EGFP in example 3 of the present invention;

FIG. 6 is a graph showing the effect of fluorescence on the expression site of EGFP localized by the endoplasmic reticulum in example 3 of the present invention;

FIG. 7 is a graph showing the results of fluorescence effects of the expression sites of EGFP localized extracellularly in example 3 of the present invention;

FIG. 8 is a vector map of co-expression of the EXO gene and the IL10 gene in example 4 of the present invention;

FIG. 9 is a Western blot of tobacco BY-2 cells expressing IL10 of example 4 of the present invention, taken sequentially from left to right: intracellular extract (Cell) and medium (Media) of non-transgenic tobacco BY-2 Cell, marker (see right figure for specific size), IL10 standard (Std), intracellular extract (Cell) and medium (Media) under IL10 and EXO coexpression condition;

FIG. 10 is a map of an expression vector of a herceptin antibody in example 5 of the present invention; wherein HC is the coding region of the Herceptin heavy chain; LC is the herceptin light chain coding region; hygR is a hygromycin resistance screening marker gene;

FIG. 11 is a Western blot of tobacco BY-2 cells expressing herceptin antibody in example 5 of the present invention; from left to right in sequence: human IgG standard (Std), marker (see right drawing for details), intracellular extract (CK) of non-transgenic tobacco BY-2 cells, intracellular extract (Cell) and Medium (Medium) under the condition of co-expression of herceptin and EXO.

Detailed Description

The technical solution of the present invention is further explained with reference to the drawings and the embodiments.

Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: coldSpringHarbor laboratory Press, 1989), or according to the manufacturer's recommendations. Both the pYBA1132 and pCAMBIA1302 vectors were purchased from Wuhan Bio-technology GmbH (28156569).

Definition and description:

the term "encoding" as used herein in the context of a particular nucleic acid means that the nucleic acid contains the necessary information to direct translation of the nucleotide sequence into a particular protein.

The term "promoter" in the present invention refers to a nucleic acid molecule, which is usually located upstream of the coding sequence of a gene of interest, provides a recognition site for RNA polymerase, and is located upstream in the 5' direction of the transcription initiation site of mRNA. It is an untranslated nucleic acid sequence to which RNA polymerase binds to initiate transcription of a gene of interest. In ribonucleic acid (RNA) synthesis, a promoter may interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription), including the core promoter region and regulatory regions, like a "switch," which determine the activity of the gene and, in turn, control the initiation of protein production by the cell. Exemplary promoters useful in the present invention include any of the CaMV35S promoter, enhanced CaMV35S promoter, NOS promoter, ACTIN promoter.

The term "expression" in the context of the present invention includes any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The expression "EXO gene" in the invention refers to the coding gene of the Arabidopsis thaliana extracellular subunit EXO70 family protein E2, including but not limited to the naturally occurring Arabidopsis thaliana extracellular subunit EXO70 family protein E2 gene, and the EXO gene mutant which is artificially modified and still has the EXO70 family protein E2 activity.

"expression vector" refers to a DNA construct used to express a polypeptide encoding a desired polypeptide. Recombinant expression vectors can include, for example, vectors comprising 1) a collection of genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; 2) A structural or coding sequence that is transcribed into mRNA and translated into protein; and 3) transcription subunits of appropriate transcription and translation initiation and termination sequences. The expression vector is constructed in any suitable manner. Any plant overexpression vector known in the art can be used as the vector used in the present invention, including pCAMBIA series, pEarleyGate series, pGreenII series, etc.

Illustratively, the vector designed by the invention is a characterization plasmid pYBA1132-cytEGFP, pYBA1132-sseEGFP and pYBA1132-sseEGFP-KDEL which is constructed based on pYBA1132 plasmid and has an expression regulation sequence, and the plasmid map is shown in figure 2. T-DNA left border in FIG. 2 represents the left border sequence of the T-DNA region; NOS promoter means NOS promoter; nptII is a screening marker gene used for coding neomycin phosphotransferase; NOS terminator represents an NOS terminator; caMV35S promoter means CaMV35S promoter; atCOR47 ' 5' UTR represents a 5' untranslated region; EGFP is a fluorescent label and is used for coding enhanced green fluorescent protein; 35S polyA can also be written as CaMV poly-A signal, representing a terminator; T-DNA right border denotes the right border sequence of the T-DNA region; atEXO is a sequence encoding EXO; ntPR1a-SSP is a sequence for coding a secretory signal peptide; KDEL is a sequence encoding the endoplasmic reticulum retention signal.

The term "transformation" in the present invention has a meaning generally understood by those skilled in the art, i.e., a process of introducing exogenous DNA into a host. Methods of transformation include any method of introducing nucleic acid into a cell, including, but not limited to, biolistic methods, protoplast transformation methods, microinjection methods, and agrobacterium-mediated methods.

Example 1 construction of EXO and different cell-localized EGFP Co-expression vectors

1. Construction of EGFP expression vector for different cell positioning

The company (general purpose organism, china) synthesized the sequences of the polyclonal restriction sites BamHI and XhoI, the 35S polyA terminator and the CaMV35S promoter; then cutting the pYBA1132 vector into linearity by KpnI enzyme; the synthesized fragments are connected through a seamless connection kit (Novozan, china) to obtain a pYBA1132 expression vector, and the map of the expression vector is shown in figure 1.

The nucleic acid sequence of NtPR1a-SSP, atCOR47 'UTR and KDEL sequence are synthesized by entrusted companies (Universal Bio, china), the nucleic acid sequence of NtPR1a-SSP is shown as SEQ ID NO.2, and the nucleic acid sequence of AtCOR 47' UTR is shown as SEQ ID NO.3. The synthetic fragment AtCOR 47' UTR was subjected to homologous recombination (Novozam, china) and ligated into a linearized pYBA1132 expression vector digested with KpnI (Takara, japan) and purified (Promega, USA), to finally obtain pYBA1132-cytEGFP (cytoplasm-localized EGFP expression vector), and the vector map is shown as A in FIG. 2. AtCOR47 ' 5' UTR and NtPR1a-SSP were ligated into the 5' end of EGFP gene in a linearized pYBA1132 expression vector digested and purified with KpnI by a single-step method (Novosa, china) to obtain pYBA1132-sseEGFP (extracellular EGFP expression vector), and the vector map is shown as B in FIG. 2. KDEL sequence is introduced into the 3' end of EGFP gene in pYBA1132-sseEGFP expression vector through a point mutation kit (Biyuntian, china). Thus, pYBA1132-sseEGFP-KDEL (EGFP expression vector localized to the endoplasmic reticulum) was obtained, and the vector map was as shown by C in FIG. 2.

2. Construction of EGFP co-expression vector for EXO and different cell positioning

The company (general organisms, china) synthesized an EXO gene having a nucleotide sequence of SEQ ID NO.1 and added restriction sites for restriction endonucleases BamHI and XhoI at both ends of the synthesized EXO gene; three different cell-localized EGFP expression vectors prepared in step 1 were double-digested with BamHI and XhoI (Takara, japan; digestion system: 10 XBuffer: 2. Mu.L, bamHI: 0.5. Mu.L, xhoI: 0.5. Mu.L, EGFP expression vector: 5. Mu.L, sterilized water: 12. Mu.L), double-digested at 37 ℃ for 1 hour, and linearized plasmids (Promega, USA) were column-purified. The purified plasmid was ligated with EXO gene (Takara, japan; ligation system: 10 XBuffer: 0.5. Mu.L, EGFP expression vector after digestion: 2. Mu.L, EXO gene after digestion: 0.5. Mu.L, T4 ligase: 0.5. Mu.L, sterile water: 1.5. Mu.L) by T4 ligase at 16 ℃ overnight. The ligation product was transformed into E.coli. Positive clones were selected on kanamycin plates and the quality-improved pellets were subjected to PCR and sequencing validation. The sequencing result is correct, three co-expression vectors are obtained, namely pYBA1132-EXO-cytEGFP, pYBA1132-EXO-sseEGFP and pYBA1132-EXO-sseEGFP-KDEL, and maps of the three co-expression vectors are shown in figure 3.

Example 2 genetic transformation of tobacco BY-2 cells

1. Agrobacterium transformation

The six expression vectors prepared in example 1 were transformed into agrobacterium tumefaciens LBA4404 (agrobacterium for short) by a heat shock method, and positive clones were screened and verified by kanamycin, streptomycin and rifampicin. The method comprises the following specific steps:

removing Agrobacterium tumefaciens LBA4404 competent cells (7 tubes each with 50 uL) from a-80 deg.C refrigerator, and thawing on ice for 5min; adding 500ng of plasmids (6 plasmids constructed above) into 50uL of Agrobacterium infected cells, making 6 tubes, sucking, mixing, and standing on ice for 30min; transferring into liquid nitrogen and standing for 5min; placing in water bath at 37 deg.C for 5min; standing on ice for 5min; respectively adding 800uLYEB liquid culture medium (without antibiotics), and placing on a shaking table with 28 deg.C and rotation speed of 200rpm for 3h; centrifuging at 5000rpm for 5min, and discarding the supernatant (leaving a small amount of culture solution); appropriate amounts of each were smeared on YEB plates containing kanamycin sulfate and rifampicin, and cultured at 28 ℃ for 2 days.

2. The recombinant agrobacterium is used for transforming the tobacco BY-2 cells.

100mL of YEB broth was taken and 40uL of rifampicin at 50mg/mL and 100uL of kanamycin sulfate at 50mg/mL were added. The transformed Agrobacterium tumefaciens LBA4404 of step 1 was taken out from the freezer at-80 ℃ and inoculated into the above medium. The cells were incubated at 28 ℃ in an incubator at 200 rpm. After the OD value of each tube of bacterial liquid is about 0.8-1.2, centrifuging at 6000rpm for 10min by using a centrifuge, discarding the supernatant, re-suspending with MS culture solution until the OD value is about 3, and adding acetosyringone with the final concentration of 100 mu M. 5mL of tobacco BY-2 cells cultured for four days were pipetted into a petri dish having a diameter of 9cm, and 200. Mu.L of the above-mentioned resuspended solution was added thereto. After mixing gently, the cells were cultured in the dark for 3 days.

3. And selecting an MS solid culture medium plate added with 50mg/L kanamycin and 200mg/L cefamycin to screen the transgenic tobacco BY-2 cells. Selecting the micro callus with higher fluorescence intensity for continuous subculture, and then transferring the micro callus into a liquid MS culture medium for suspension culture, wherein the culture conditions are as follows: 25 ℃,120rpm, dark. Subculturing every seven days, wherein the subculturing proportion is 10% (v/v).

The results show that we obtained transgenic tobacco BY-2 cells BY Agrobacterium-mediated genetic transformation, as shown in FIG. 4. Transgenic calli with stronger fluorescence intensity were visible under fluorescence microscope (fig. 4 a). After suspension culture of the callus, it was seen that most cells had significant green fluorescence (FIG. 4 b).

Example 3 Effect of Co-expression of EXO on the different localization of EGFP expression sites

And (3) sucking the transgenic tobacco BY-2 cells cultured for 4 days onto a glass slide, and observing the subcellular localization of the EGFP BY a laser confocal microscope. The results of the experiment are shown in FIGS. 5-7.

From the results, it can be seen that: through fluorescent observation, when only EGFP is expressed, three different expression positions are obtained by different vectors. When expressed in the cytoplasm, the green fluorescence signal comes from the cytoplasm, and no fluorescence is present in the central vacuole. The green fluorescence clearly shows the shape of the intracellular reticulon after addition of the endoplasmic reticulum retention signal in the coding region. EGFP added with secretory signal peptide is distributed in all cells, and the cells emit green color throughout. When co-expressed with EXO genes, EGFP localized in the endoplasmic reticulum and extracellularly apparently aggregated, appearing as very many bright small dots within the cell. For the cytosolic EGFP, although a small number of cells showed aggregation, most of the fluorescence remained localized in the cytoplasm, with a similar dispersion to the control (when EGFP alone was expressed).

The above results indicate that EXO over-expression forms an EXO organelle that is not only capable of transporting proteins in the cytoplasm, but also has a loading function for proteins localized to the endoplasmic reticulum and proteins localized to the secretory pathway (localized to the extracellular space), and the packaging of the former (localized to the cytoplasm) may be much less than the latter two (localized to the endoplasmic reticulum and the extracellular space). This is probably because EXO recruits proteins located in the endoplasmic reticulum when it accumulates in the endoplasmic reticulum intima and adventitia, and when an EXO organelle is formed, these recruited proteins are encapsulated and appear to aggregate.

Based on the results, the EGFP secretion rate and the total yield of the tobacco BY-2 cells were determined BY the inventors. The results of the experiment are shown in table 1.

Table 1 effect of overexpression of EXO genes on EGFP protein secretion rate and total yield at different expression sites.

* : significant differences from control at 95% confidence level; * *: there was a significant difference from the control at the 99% confidence level.

From the results, the secretion rate and the total yield of EGFP at three expression positions are improved by over-expressing EXO, but the secretion rate and the total yield of EGFP localized to the endoplasmic reticulum and extracellularly are improved more remarkably, and especially the secretion rate and the total yield of EGFP localized to the endoplasmic reticulum are improved most remarkably. This is probably because EGFP secreted extracellularly not only reaches the extracellular but also reaches the vacuole, which contains a large amount of proteases and has a degrading effect on EGFP.

EXAMPLE 4 extracellular secretion of recombinant protein human Interleukin 10

1. Construction of expression vector of recombinant protein human interleukin 10

A DNA molecule having a nucleic acid sequence of SEQ ID NO.4 was synthesized by a company (general purpose organism, china), and AtCOR47 '5' UTR and a gene Ntpr1a-SSP (SEQ ID NO. 2) encoding a secretion signal peptide were added to the 5 'end of the synthesized DNA molecule, and a gene encoding an endoplasmic reticulum retention signal KDEL was added to the 3' end thereof; and the restriction endonuclease cut sites KnpI and NruI are respectively added at the two ends. The pYBA1132 expression vector in the embodiment 1 is subjected to KnpI and NruI double enzyme digestion (Takara, japan) and then is connected with the synthesized artificial sequence through homologous recombination (NuoZan, china) to obtain a recombinant vector pYBA1132-IL10; the pYBA1132-IL10 expression vector was subjected to double digestion with BamHI and XhoI (Takara, japan), followed by ligation with the EXO gene synthesized in example 1 by homologous recombination (Novozam, china), to obtain a recombinant vector pYBA1132-IL10-EXO (1132-IL 10-EXO), the results of which are shown in FIG. 8.

2. Tobacco BY-2 cell genetic transformation

The recombinant vectors pYBA1132-IL10 and pYBA1132-IL10-EXO are respectively transformed into tobacco BY-2 cells, and the specific method is the same as that in example 2.

3. Qualitative and quantitative determination of recombinant protein IL10

1.5mL of the tobacco BY-2 cells suspended in step 2 was pipetted into a 2mL centrifuge tube and centrifuged at 3000rpm for 5min. The supernatant medium was aspirated, and the lower cells were added with a protein extract (20 mM tris-HCl buffer pH 8.0, 10mM EDTA,0.2% Triton X-100, 10mM PMSF), and the cells were disrupted by a hand-held disrupter. The extract was centrifuged at 8000rpm for 5min and the supernatant was collected. IL10 was characterized by Western blotting, with rabbit anti-human IL10 monoclonal antibody (Cat: 10947-R001, nano Biological, china) as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit antibody (A0208, bilun day, china) as the secondary antibody. The quantification of IL10 was performed according to the enzyme-linked immunosorbent assay (ELISA) KIT (Cat: KIT10947A, nano Biological, china). The results are shown in fig. 9 and table 2.

TABLE 2 Effect of overexpression of EXO Gene on IL10 protein secretion Rate and Total yield

* *: there was a significant difference from the control at the 99% confidence level.

The results show that the antibody detects the standard human IL10 protein at 21.9kDa, whereas in cell extracts the antibody binds significantly to the membrane at slightly below 45 kDa. Since IL10 protein is often expressed in tobacco as a dimer, the above experimental results confirmed that IL10 protein is indeed expressed in transgenic tobacco BY-2 cells. As can be seen from table 2, the secretion rate and total yield of IL10 were significantly improved by overexpression of the EXO gene.

Example 5 extracellular secretion of Herceptin antibodies

1. Construction of expression vector for Herceptin antibody

The company (general purpose organism, china) synthesizes a herceptin heavy chain DNA molecule with a nucleic acid sequence of SEQ ID NO.5 and a herceptin light chain DNA molecule with a nucleic acid sequence of SEQ ID NO.6, then adds AtCOR47 ' UTR and a gene NtPR1a-SSP coding a secretion signal peptide at the 5' ends of the synthesized herceptin heavy chain DNA molecule and the synthesized herceptin light chain DNA molecule, and adds a gene coding an endoplasmic reticulum retention signal KDEL at the 3' ends; and SpeI and BstEII enzyme cutting sites are added at both ends of the light chain. The pCAMBIA1302 vector was subjected to SpeI and BstEII double enzyme digestion (Takara, japan), and then ligated to the light chain DNA molecule synthesized as described above by homologous recombination, to obtain an expression vector pCAMBIA1302-LC. Both ends of the heavy chain were introduced into the CaMV35S promoter and CaMV polyA terminator (Novozan, china) by homologous recombination. EcoRI sites and HindIII sites were introduced at both ends of the heavy chain. After EcoRI and HindIII double enzyme digestion (Takara, japan) is carried out on the pCAMBIA1302-LC vector, the pCAMBIA1302-LC vector is connected with heavy chain DNA molecules through homologous recombination, and finally, a recombinant vector pCAMBIA1302-LH is obtained, wherein the vector map is shown in figure 10. Meanwhile, the pYBA1132-EXO-cytEGFP vector in example 1 was used as an expression vector for EXO.

2. Tobacco BY-2 cell genetic transformation

The recombinant vectors pCAMBIA1302-LH and pYBA1132-EXO-cytEGFP are respectively transformed into agrobacterium tumefaciens, and the specific transformation method is the same as that in example 2.

The two kinds of agrobacterium are mixed according to the proportion of 1. Hygromycin and kanamycin were screened for positive calli. Carrying out suspension culture on the callus, wherein the culture conditions are as follows: 25 ℃,120rpm, dark. Subculturing every seven days, wherein the subculturing proportion is 10% (v/v).

3. Qualitative and quantitative determination of herceptin antibodies

The sample preparation procedure was the same as example 4, step 3. The herceptin antibodies were characterized by western blotting. Horseradish peroxidase-conjugated goat anti-human antibody (A18853, sigma, USA) was diluted 1000-fold with diluent and incubated overnight at 4 ℃ for 16 hours. After incubation was complete, 5 washes with TBST were added for 5 minutes each. And uniformly mixing 500 mu L of developing solution BeyoECL Plus A and BeyoECL Plus B (in Biyun, china) by using a liquid transfer gun, uniformly dripping the mixture on a film, and exposing and observing the film by using a gel imager. Quantification of herceptin was performed according to ELISA kit instructions (ab 195215, abcam, USA). The results are shown in fig. 11 and table 3.

TABLE 3 Effect of overexpression of EXO Gene on Herceptin secretion and Total yield

The result shows that the heavy chain protein and the light chain protein of the IgG antibody are respectively detected at 50kDa and 25kDa in the tobacco cell extract and the culture medium, and the fact that the Herceptin antibody is expressed in the transgenic tobacco BY-2 cell is confirmed. The secretion rate and total yield of herceptin were significantly improved by overexpression of EXO gene.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

SEQUENCE LISTING

<110> Hunting (Jiaxing) Biotech Ltd

<120> a method for increasing extracellular secretion of recombinant protein in plant cells

<130> JX202110029

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1920

<212> DNA

<213> Artificial sequence

<400> 1

atggcagagt ttgattccaa ggttcctgtt tccggaatga acaatcatgt ctttgaggca 60

tgtcaccatg ttgttaaggc actgagggca tctgataaca acctggatgc caatttgaga 120

aagctcctaa gtgacctaga gatgcatttg tcaacatttg ggattgctga taccaaagtt 180

gaagatgcag gattctctga gatcaagaag agattcaaag aagctgtgaa gagaatccgc 240

agttgggaga caaaccagtc gacgatgttt gaagctggtc tgtctgaagc tgatcagttc 300

tttcaagccc tgtatgacgt tcaaacagtt cttgtgggtt tcaaagcttt gcccatgaaa 360

actaaccaga tggagaaaga tgtttacaac caagctacgg ttgctcttga catcgcgatg 420

ttgaggcttg agaaagagct ctgtgatgtt ctgcatcagc acaaacggca tgtacagcct 480

gattacttgg ctgttagttc ccgtagaaaa gatattgtgt acgacgagtc ctttgtctcc 540

ctagatgatg aagttattgt cgaagcttct tcacatgaag atgatgaaca gatatcggac 600

ttttataatt ctgatttggt tgatcccatc gtacttcctc acatcaaagc tattgcaaac 660

gccatgtttg cttgcgagta tgatcaacca ttctgtgaag ctttcatcgg cgttcaaaga 720

gaagctctgg aggagtatat ggttactctc gaaatggaaa gattcagctg cgttgatgtt 780

ctcagaatgg actgggagga tttgaatggt gcaatgagaa aatggacaaa ggttgttaag 840

atcattactc aggtctatct cgccagtgaa aaacagctat gtgatcagat tttgggagac 900

tttgagtcaa tttctacagc ttgcttcatt gaaatctcaa aagatgcaat tctatcactc 960

ctcaactttg gagaagctgt ggtgctgaga tcttgcaagc cagaaatgct tgagcgcttc 1020

cttagtatgt atgaggtttc agcagagatt ctcgtggatg tcgataacct cttccccgat 1080

gaaacaggct cgtctttaag aattgcattt cacaacctgt caaaaaaact agctgatcat 1140

acaaccacaa ccttcttaaa gttcaaagac gcaatagcct cagatgaatc cacacgcccc 1200

tttcatggag gcgggattca tcacctgacc aggtatgtaa tgaactactt gaagcttctc 1260

ccggagtata ccgactcact aaactcactg cttcagaaca tacacgttga cgactccatc 1320

cccgagaaaa caggagaaga tgtcctccct tcgacattct ctccaatggc tagacacctc 1380

aggtcaatcg tcacaactct agaatccagc cttgagcgaa aagctcagct gtacgcagac 1440

gaagcactga aatccatttt cctgatgaac aacttccgtt acatggtcca gaaggtaaag 1500

ggatcagagc tgagacgctt gtttggagac gaatggatca ggaagcacat tgcaagctac 1560

caatgcaatg tcactaacta cgagagatcc acatggagct ccatactcgc tttgctcaga 1620

gacaacaacg actctgtaag aaccctgaga gaaagatgca gacttttcag ccttgcattt 1680

gatgatgttt acaagaacca aacccgttgg tcagtccctg attcagagct ccgcgatgat 1740

cttcacatct cgacctctgt caaggttgtt cagtcttaca gaggatttct tggaagaaat 1800

gcagttcgca taggcgaaaa acacatccga tacacttgtg aagacattga gaatatgctc 1860

cttgatctct ttgaatgttt gccatctcca agatcactgc gcagctctcg taagagatga 1920

<210> 2

<211> 90

<212> DNA

<213> Artificial sequence

<400> 2

atgggatttg ttctcttttc acaattgcct tcatttcttc ttgtctctac acttctctta 60

ttcctagtaa tatcccactc ttgccgtgcc 90

<210> 3

<211> 107

<212> DNA

<213> Artificial sequence

<400> 3

caaacattac tcattcacaa aaccatctta aagcaactac acaagtcttg aaattttctc 60

atattttcta tttactatat aaacttttaa tcaaatcaag attaaag 107

<210> 4

<211> 537

<212> DNA

<213> Artificial sequence

<400> 4

atgcacagct cagcactgct ctgttgcctg gtcctcctga ctggggtgag ggccagccca 60

ggccagggca cccagtctga gaacagctgc acccacttcc caggcaacct gcctaacatg 120

cttcgagatc tccgagatgc cttcagcaga gtgaagactt tctttcaaat gaaggatcag 180

ctggacaact tgttgttaaa ggagtccttg ctggaggact ttaagggtta cctgggttgc 240

caagccttgt ctgagatgat ccagttttac ctggaggagg tgatgcccca agctgagaac 300

caagacccag acatcaaggc gcatgtgaac tccctggggg agaacctgaa gaccctcagg 360

ctgaggctac ggcgctgtca tcgatttctt ccctgtgaaa acaagagcaa ggccgtggag 420

caggtgaaga atgcctttaa taagctccaa gagaaaggca tctacaaagc catgagtgag 480

tttgacatct tcatcaacta catagaagcc tacatgacaa tgaagatacg aaactga 537

<210> 5

<211> 1431

<212> DNA

<213> Artificial sequence

<400> 5

atgaactttg tgctcagctt gattttcctt gccctcattt taaaaggtgt ccagtgtgaa 60

gttcaacttg ttgagtcagg aggtggattg gttcaaccag gtggatctct tagattgtca 120

tgtgctgctt ctggttttaa tattaaggat acttacatcc attgggttag acaagctcca 180

ggtaaaggac ttgagtgggt tgctaggatc tatcctacta atggttatac aagatacgct 240

gattctgtta agggaaggtt tactatctca gctgatactt ctaaaaatac agcttatctt 300

cagatgaact ctttgagagc tgaagataca gctgtttatt actgttctag atggggtgga 360

gatggatttt atgctatgga ttactggggt caaggaactc ttgttacagt ttcttcagct 420

tcaactaagg gtccatctgt ttttccattg gctccttctt caaaatcaac ttctggtgga 480

acagctgctc ttggatgctt ggttaaggat tactttccag agcctgttac agtttcttgg 540

aactctggtg ctcttacttc aggagttcat acatttcctg ctgttttgca atcttcaggt 600

ctttattctt tgtcttcagt tgttactgtt ccatcttcat ctttgggaac tcaaacatac 660

atctgtaacg ttaaccataa gccttcaaac acaaaggttg ataagaaggt tgagccacct 720

aagtcttgcg ataaaactca tacatgtcca ccttgcccag ctcctgaact tttgggtgga 780

ccatctgttt ttctttttcc acctaagcct aaagatactt tgatgatttc aagaactcca 840

gaggttacat gtgttgttgt tgatgtttct catgaagatc ctgaggttaa gtttaattgg 900

tatgttgatg gtgttgaagt tcataatgct aagactaaac caagagaaga gcaatataat 960

tcaacttaca gggttgtttc tgttcttaca gttttgcatc aagattggct taatggtaaa 1020

gaatacaagt gtaaagtttc aaataaggct ttgccagctc ctatcgaaaa gactatctct 1080

aaggctaaag gacaaccaag agagcctcaa gtttatacac ttccaccttc aagggatgaa 1140

ttgactaaga accaagtttc tcttacatgc ttggttaaag gtttttaccc atcagatatt 1200

gctgttgaat gggagtctaa tggtcaacct gagaataact ataagactac accacctgtt 1260

cttgattctg atggttcttt ctttctttac tcaaagttga ctgttgataa gtctaggtgg 1320

caacaaggaa atgttttctc ttgctctgtt atgcatgaag ctcttcataa ccattacaca 1380

caaaagtcac tttctttgtc acctggaaag tctgaaaaag atgagttgtg a 1431

<210> 6

<211> 702

<212> DNA

<213> Artificial sequence

<400> 6

atgaactttg tgctcagctt gattttcctt gccctcattt taaaaggtgt ccagtgtgat 60

attcaaatga ctcaatcacc atcttcactt tctgcttcag ttggagatag agttactatt 120

acatgtaggg cttctcaaga tgttaataca gctgttgctt ggtatcaaca aaagccagga 180

aaagctccta agttgttgat ctattctgct tcatttttgt actcaggtgt tccatctaga 240

ttttctggat caaggtctgg tactgatttc actcttacaa tctcttcatt gcaacctgaa 300

gatttcgcta catactactg tcaacaacat tacactacac cacctacttt cggacaaggt 360

acaaaggttg aaattaagag aactgttgct gctccatcag tttttatttt tccaccttct 420

gatgagcaac ttaaatcagg aacagcttct gttgtttgcc ttttgaataa cttctaccct 480

agggaggcta aagttcagtg gaaggttgat aatgctcttc aatctggtaa ttctcaagaa 540

tcagttactg agcaagattc taaggattca acatactctt tgtcttcaac tcttacattg 600

tcaaaggctg attacgaaaa gcataaggtt tacgcttgtg aggttactca tcaaggattg 660

tcttcacctg ttacaaagtc ttttaataga ggtgaatgct ga 702

Claims

1. A method of increasing extracellular secretion of a recombinant protein in a plant cell, the method comprising the steps of:

2) Transforming a plant cell to secretionally express a recombinant protein;

wherein the nucleic acid sequence of the EXO gene is shown as SEQ ID NO.1, and the nucleic acid sequence of the gene for coding the recombinant protein comprises a nucleic acid sequence for coding a secretory signal peptide connected to the N end of the recombinant protein; the secretory signal peptide is NtPR1a-SSP, and the nucleic acid sequence for coding the NtPR1a-SSP is shown as SEQ ID NO. 2; the nucleic acid sequence of the gene encoding the recombinant protein comprises a nucleic acid sequence encoding an endoplasmic reticulum retention signal linked to the C-terminus of the recombinant protein, wherein the endoplasmic reticulum retention signal is KDEL or HDEL; the recombinant protein is interleukin 10 or herceptin antibody; the plant cell is a tobacco suspension cell or an arabidopsis thaliana suspension cell, the KDEL is a Lys-Asp-Glu-Leu amino acid residue sequence, and the HDEL is a His-Asp-Glu-Leu amino acid residue sequence.

2. A method of increasing extracellular secretion of a recombinant protein by a plant cell according to claim 1, wherein: the 5 'end of the nucleic acid sequence of the gene for coding the recombinant protein is connected with a non-coding region, the non-coding region is AtCOR 47' UTR, and the nucleic acid sequence is shown as SEQ ID NO.3.

3. A method of increasing extracellular secretion of a recombinant protein by a plant cell according to claim 1, wherein: the EXO gene or the gene of the coding recombinant protein is driven by a promoter, and the promoter is a CaMV35S promoter, an enhanced CaMV35S promoter, an NOS promoter or an ACTIN promoter.