CN101792934A - Novel method for building ultra-high capacity gene library based on combination principle and PCR - Google Patents

Abstract

Directed evolution of enzymes is an effective way to change the properties of enzymes and improve their utilization value. However, existing directed evolution methods all rely on existing genes and can only be modified on the basis of discovered genes. Therefore, nothing can be done for screening targets that do not exist in nature or that appear newly. The invention utilizes the random fragments with a common joint, connects, combines and reassembles the random fragments by PCR method, and obtains a huge gene library of random combination. This library theoretically contains any gene sequence, which can be used to screen any target traits, so that new genes can be created, breaking through the limitation that the original directed evolution method can only modify existing genes. The present invention (establishing a super-large-capacity gene library) will make directed evolution and drug body screening no longer be troubled by the limited diversity of the gene library, and provide a huge resource bank for screening, and some targets that are difficult to screen for existing gene libraries, such as The screening of new target receptors, the discovery of unnatural conditional enzymes, and the detection of new target substances play an active role.

Description

A New Method for Establishing Gene Library with Large Capacity Based on Combinatorial Theory and PCR

technical field

The invention relates to a method for establishing a gene library in molecular evolution, using random fragments with shared joints, and performing complementary, overlapping extension and reunion on the random fragments by means of PCR to obtain a randomly combined huge gene library.

Background technique

As biocatalysts, enzymes have the advantages of high catalytic efficiency, strong specificity, mild reaction conditions, and environmental friendliness. The U.S. Department of Energy, the Department of Commerce and other departments predict that biocatalysts will become a necessary tool for the sustainable development of the chemical industry in the 21st century. It is the core of industrial biotechnology, but the properties of natural enzymes are difficult to adapt to various industrial production conditions. Directed evolution of enzymes (Directed Evolution, DE) is an effective way to change the structure and properties of enzymes to meet this demand.

Directed evolution of enzymes (or proteins), also known as In vitro Molecular Directed Evolution of enzymes (In vitro Molecular Directed Evolution), is a process of simulating Darwinian evolution in the laboratory, using improved mutagenesis techniques combined with recombination methods to establish mutant libraries and create Molecular diversity, combined with sensitive high-throughput screening technology, can quickly obtain ideal non-natural enzymes (or cycle into the next round of directed evolution in order to obtain more ideal non-natural enzymes). Directed evolution is different from the rational design of proteins. It does not need to know the protein structure, conserved sites, catalytic mechanism, etc. in advance, and compared with natural evolution, the evolution time is reduced to months or even weeks. Directed evolution technology has been used in the evolution of hundreds of enzymes, greatly improving the activity and efficiency of biological enzymes, and has had a huge impact on the production of industry, agriculture, and medicine.

Directed evolution of enzymes requires two important supporting technologies: one is to establish a mutant library with a sufficient amount of variants to provide abundant materials for further screening; the other is to have a suitable screening system that can quickly select from many The protein that meets the target is screened from the mutant protein. The key to establishing a mutant library is to create gene diversity, and error-prone PCR (Error-prone PCR) and DNA recombination (DNARecombination) are the main existing methods. Error-prone PCR is to randomly introduce mismatched bases into corresponding DNA molecules by changing the concentration of Mg ²⁺ and dNTPs in the reaction system to obtain random mutants of enzyme molecules, and then undergo multiple such small-step iterative mutations (1 -2 amino acids), the desired enzyme function is obtained by sequentially or combinatorially accumulated mutations. Error-prone PCR has certain limitations in practical application: first, the mutation speed is slow; second, only point mutations are introduced; third, the gene fragments involved are too short (less than 800bp). Another major method of generating mutations is the random splicing or family rearrangement of homologous genes in vivo. At present, there are many methods of DNA recombination, such as DNA shuffling (DNA Shuffling), staggered extension PCR (StEP), exon DNA random recombination, random primer in vitro recombination (RPR), incremental cleavage method to generate hybrid enzyme (ITCHY), Random insertion and deletion (RID), these methods differ from error-prone PCR in that DNA recombination relies on mixing and ligating numerous fragments of parental DNA to create mutants, known as sexual PCR. Among the many DNA recombination methods, DNA shuffling has been widely used. The basic principle is: firstly, a group of homologous genes or mutant gene fragments in the gene bank are obtained by means of PCR or enzyme digestion, and then chemical (DNaseI ) or physical (ultrasonic) means to randomly cut it into small fragments within a certain length range, because these small fragments have a certain degree of homology between each other as primers, through re-assembly PCR (Re-assembly via PCR) Pathways were extended to have full-length gene segments. Template shifting occurs when small fragments from one copy of the gene prime each other with small fragments from the other copy. This method can not only create the opportunity to combine the mutations in the parental gene group as much as possible to obtain mutants that lead to greater variation, but also integrate the advantages of the paternal genes.

Although DNA shuffling and its derivative techniques are powerful tools for obtaining beneficial mutants, they are generally not applicable to recombining sequences with less than 70%-80% homology. There are many kinds of proteins in nature, which have different sequences (low homology) and similar spatial structures. In order to recombine these proteins, Benkovic's research group established a method of incremental cleavage to generate hybrid enzymes (ITCHY) to generate DNA-independent Recombinant libraries of sequence homology. Sieber et al. proposed a protein recombination (SHIPREC) method that does not depend on sequence homology (SHIPREC). Random fragments of single gene length were recovered by agarose gel electrophoresis, which ensured the conservation of the length of the progeny chimera and maintained a proper sequence match for the crossover. , mainly occur at structurally related sites, and the two amino acids at the intersection are still in their positions in the parental protein structure, thereby increasing the proportion of functional heterozygotes in the library. Nonhomologous random recombination (NRR, nonhomologous random recombination) method proposed by Liu et al. can randomly recombine two non-homologous parental genes through random connection of ligase.

The existing methods of establishing mutant libraries have greatly promoted the evolution of enzymes, especially the production of biocatalysts suitable for pharmaceutical and industrial production, but these methods can only rely on existing genes for modification and screening, and are difficult to develop In the new sequence space (especially when the function changes greatly), for screening targets that do not exist in nature, such as the prevention of emerging viruses or the screening of unnatural traits, it is difficult to use the original gene transformation to achieve.

Therefore, by designing random sequences, using the combination principle and PCR method, the present invention intends to establish a method for constructing a super-large-capacity gene library, which does not depend on the existing gene library and can theoretically contain the sequence space encoding any protein .

Contents of the invention

The purpose of the present invention is to provide a method for establishing a gene library.

The method provided by the present invention is called the method for establishing a gene library based on the combination principle and PCR, comprising the following steps:

1. Design a random DNA or RNA sequence with a common linker sequence at both ends of the sequence.

2 PCR recombination of random sequences in solution using the complementarity of the shared linker sequence.

3 Add specific primers containing the linker sequence in 1 to control the length of the recombination product during the recombination process.

4 Amplify the recombination product with specific primers by means of PCR.

5. Perform asymmetric PCR on the DNA or RNA product in step 2 or 3 or 4 to obtain single-stranded nucleic acid fragments, and perform screening to obtain DNA molecules or RNA molecules that recognize and specifically bind to the target substance.

6. Recover the fragments of the DNA products in step 2, 3 or 4, and screen to obtain proteins that recognize and specifically bind to the target substance or proteins with any other functions by means of enzyme digestion, ligation, and transformation.

Principle of the present invention is as shown in Figure 1:

Take the target molecule of evolution as protein (enzyme) as an example: design two random sequences with a length of 20-200 bases (according to the actual selection) as the basic modules of the experimental combination (this description uses 39 bases as an example) , the two ends of the random sequence have a common linker of nine bases, and these nine bases are forward complementary, as a common connection interface, and serve as primers for each other during PCR recombination. The nine bases are selected to encode a base sequence that can form a flexible amino acid (linker). The 21 bases in the middle of the random sequence are random, encoding 7 amino acids, which is enough to form a protein motif (motif). Therefore, random sequences can be reassembled by PCR to obtain a protein that encodes alternating random motifs and flexible linkages. After repeated reunions, the reunited fragments can be continuously extended. In order to prevent the reunion fragment from being too long, after the reunion PCR reaches a certain number of cycles, a certain proportion of termination sequence is added to the reaction system. Since the termination sequence is a specific sequence, after the reunion is extended to this sequence, it will no longer Complementation and repolymerization PCR reactions are terminated. And use specific primers to amplify the sequence after recombination to ensure the diversity of the gene library. Determine the size of the recovered fragments according to the screening target, and carry out enzyme digestion, ligation and transformation on the recovered gene fragments, and finally randomly select positive clones from the transformed clones for sequencing to test the diversity of the library sequence.

If the target molecule of evolution is a DNA molecule or an RNA molecule, the principle of the invention is as follows: design two random sequences (according to the actual selection) with a length of 9-300 bases, as the basic module of the experimental combination, and the two ends of the random sequence are respectively A common linker with nine bases, which are forward complementary, serves as a universal connection interface and serves as primers for each other during PCR recombination. Therefore, a DNA or RNA molecule in which a shared linker and random sequences are alternately obtained can be obtained by PCR recombination of random sequences. After repeated reunions, the reunited fragments can be continuously extended. Determine the size of the recovered fragments according to your own screening goals, and obtain single-stranded nucleic acid fragments through asymmetric PCR, identify and bind the single-stranded nucleic acid fragments and target substances after mixing, and screen according to the size of the binding force.

The method for setting up a gene library based on combination principle and PCR of the present invention has the following advantages:

1Using random fragments with shared adapters to connect, combine and recombine to obtain a huge gene library with random combinations. This library theoretically contains any gene sequence, which can be used to screen any target traits, so that new genes can be created, breaking through the limitation that the original directed evolution method can only modify existing genes. The original directed evolution method is to transform genes "from weak to strong", but the present invention is to create genes "from scratch".

2. The success of the present invention (establishment of super-large-capacity gene library) will make directed evolution and drug body screening no longer be troubled by the limited diversity of gene library, and become new screening targets, such as the detection of new viruses and the screening of new receptor targets. Provide a broad path.

3 The method is simple to establish a gene library by combining random fragments and complementation and extension of common adapters.

Description of drawings

Accompanying drawing 1 is: The schematic diagram of establishing super-large-capacity gene library based on combination principle and PCR

Accompanying drawing 2 is: Utilize random sequence repolymerization PCR product picture

Accompanying drawing 3 is: the schematic diagram of the reconstructed constitutive expression vector (pBS-SV)

PagaB is a constitutive promoter; restriction endonucleases NdeI and EcoRI are insertion sites.

Detailed ways

The following examples will help those of ordinary skill in the art to further understand the present invention, but do not limit the present invention in any form.

DNA sequences used in this experiment:

Random sequence A: 5′-GGATCGGCC(NNY) ₇ CCTAGCCGG-3′(39base)

Random sequence B: 5′-GGCCGATCC(NNY) ₇ CCGGCTAGG-3′(39base)

N: represents one of the four bases of ATCG; Y: represents only T or C; (NNY) ₇ represents repeating 7 times

CCT AGC CGG-3′(22base)Upstream primer: 5′-GG AAT TC

CCT AGC CGG-3′(22base)

CCG GCT AGG-3′(21base)Downstream primer: 5′-CCG

CCG GCT AGG-3′(21base)

Bases in italics are restriction sites, bases in bold are stop codons

Example 1: Screening of Neisseria meningitidis aptamers in a random library

Experimental steps:

1. Sequence design

The shared linkers at the 5' and 3' ends of one random sequence are 5'-GGATCGGCC-3' and 5'-CCTAGCCGG-3', respectively, and the shared linkers at the 5' and 3' ends of the other random sequence are 5'-GGCCGATCC- 3' and 5'-CCGGCTAGG-3'. The two ends of the two random sequences are complementary to each other and can be used as primers for recombination.

2. Reunion

After a series of condition optimization, the repolymerization system was finally determined as:

random sequence 1 300ng

Random Sequence 2 300ng

Upstream primer 12ng

Downstream primer 12ng

dNTPs(2.5mM) 1.4μL

10XTaqE Buffer 2μL

TaqE(2.5U/μL) 0.5U

Mg2 ⁺ (25mM) 1.2μL

Make up to 20μL with H ₂ O

The procedure for reconvergence PCR is:

72℃ 10min

3. Asymmetric PCR

Take 0.5 μL of the recombination PCR product as a template, add upstream (or downstream) primers, and perform asymmetric PCR amplification with a single primer. The goal is to obtain single-stranded DNA molecules.

The PCR reaction system is:

Purified template 30ng

Single primer (10μM) 0.4μL

dNTPs(2.5mM) 1.4μL

10XBuffer 2μL

TaqE(2.5U/μL) 0.5U

Mg2 ⁺ (25mM) 1.2μL

Make up to 20μL with H ₂ O

The PCR reaction procedure is:

94℃ 2min

72℃ 10min

4. Recovery of PCR products

1. Cut off the target band as small as possible under the ultraviolet light, put it into a pre-weighed Eppendorf tube, and weigh it again.

2. Add solution A (6MNaClO ₄ , 0.03MNaAc, pH5.2, a small amount of phenol red) at a ratio of 1:3 (glue weight: volume of solution A, mg/mL), shake to aid dissolution at 55-65°C for 5-10 minutes .

3. After the glue is completely dissolved, add 15 μL solution B (3M NaAc), and mix well.

4. Transfer the solution into a spin column, let it stand for 2 minutes, and centrifuge at 8000rpm for 1 minute.

5. Discard the liquid, add 500 μL solution C (35 mL absolute ethanol + 15 mL TE) to the spin column, and centrifuge at 8000 rpm for 1 min.

6. Discard the liquid, wash once again with 500 μL solution C, and centrifuge at 12,000 rpm for 1 min.

7. Place the spin column in the air for 5-10 minutes to remove residual ethanol.

8. Put the spin column in a new Eppendorf tube, add 20-30 μL sterile water or TE (pH 8.0), and let stand at 50°C for 2 minutes.

9. Centrifuge at 12000rpm for 1min, and the solution at the bottom of the Eppendorf tube will contain the recovered DNA fragments.

5. Aptamer screening

1. Add 10 μg of DNA library sequence to 400 μL of binding buffer, denature at 95°C for 5 minutes, rapidly cool to 4°C, add 20 μL of bacterial solution, and combine on a shaking table for 20-30 minutes.

2. Centrifuge at 10,000 rpm for 5 minutes.

3. Discard the supernatant, add 600 μL of 1× wash buffer to suspend the bacterial solution, centrifuge at 10,000 rpm for 5 min, and repeat this process 4 times.

4. Add 100 μL of deionized water and heat at 99°C for 3 minutes. After centrifugation, the nucleic acid eluted in the supernatant is purified with a kit, stored at -20°C, and used as a template for the next PCR reaction.

Purification steps:

①Put the supernatant into an Eppendorf tube

② Add DNA binding buffer by 2 times the volume

③All the mixture is transferred into the Spin column

④Centrifuge at 6000rpm for 1min, put the adsorption column into another collection tube

⑤Add 650μL Wash Buffer to the adsorption column, centrifuge at 12000rpm for 30-60s, discard the liquid in the liquid tube

⑥Repeat the previous step once

⑦Centrifuge at 12000rpm for 1min, then transfer the Spin column to a sterile Eppendorf tube

⑧ Add 50 μL of deionized water and let stand at room temperature for 1 min

⑨Centrifuge at 12000rpm for 1min to obtain the target DNA fragment

5. Perform PCR amplification and purification on the screened and purified DNA sequence using upstream primers and downstream primers to obtain an enhanced screening secondary library.

The PCR reaction system is:

Purified template 30ng

Upstream primer (10μM) 0.4μL

Downstream primer (10μM) 0.4μL

dNTPs(2.5mM) 1.4μL

10XBuffer 2μL

TaqE(2.5U/μL) 0.5U

Mg2 ⁺ (25mM) 1.2μL

Make up to 20μL with H ₂ O

The PCR reaction procedure is:

94℃ 2min

72℃ 10min

6. Repeat the above steps 1 to 5 for 10 times for repeated screening.

7. Until the aptamer specifically binding to Neisseria meningitidis is screened.

Embodiment 2: Screen the gene of resistance to kanamycin in random library with resistance screening method

1. Sequence design

The shared linkers at the 5' and 3' ends of one random sequence are 5'-GGATCGGCC-3' and 5'-CCTAGCCGG-3', respectively, and the shared linkers at the 5' and 3' ends of the other random sequence are 5'-GGCCGATCC- 3' and 5'-CCGGCTAGG-3'. On the one hand, the two ends of the two random sequences are complementary to each other and can serve as primers for reunion. On the other hand, the shared linkers all form meaningful amino acids, and the encoded amino acids belong to the flexible amino acids commonly used in the connection of protein domains, which facilitates the flexible connection of different random pattern sequences and ensures the relative structure of the protein pattern space. whole. In order to reduce the existence of stop codons in the gene sequence, the 24-30 bases in the middle of the random sequence can take the structural form of NNY (Y can be C or T), so that more meaningful sequences can be formed in the gene library.

2. Reunion

After a series of condition optimization, the recombination system was finally determined, in which a termination primer was added to control the size of the recombination fragment:

random sequence 1 300ng

Random Sequence 2 300ng

Upstream primer 12ng

Downstream primer 12ng

dNTPs(2.5mM) 1.4μL

10XTaqE Buffer 2μL

TaqE(2.5U/μL) 0.5U

Mg2 ⁺ (25mM) 1.2μL

Make up to 20μL with H ₂ O

The procedure for reconvergence PCR is:

94℃ 2min

72℃ 10min

3. Massive amplification of reunited fragments

The reaction procedure for repolymerization PCR is:

Purified template 100ng

Upstream primer (10μM) 0.4μL

Downstream primer (10μM) 0.4μL

dNTPs(2.5mM) 1.4μL

10XBuffer 2μL

TaqE(2.5U/μL) 0.5U

Mg2 ⁺ (25mM) 1.2μL

Make up to 20μL with H ₂ O

The procedure for reconvergence PCR is:

94℃ 2min

72℃ 10min

4. Recovery of PCR products

1. Cut off the target band as small as possible under the ultraviolet light, put it into a pre-weighed Eppendorf tube, and weigh it again.

2. Add solution A (6M NaClO ₄ , 0.03M NaAc, pH5.2, a small amount of phenol red) at a ratio of 1:3 (glue weight: solution A volume, mg/mL), shake at 55-65°C for 5-10 minutes Solvent.

3. After the glue is completely dissolved, add 15 μL solution B (3M NaAc), and mix well.

4. Transfer the solution into a spin column, let it stand for 2 minutes, and centrifuge at 8000rpm for 1 minute.

5. Discard the liquid, add 500 μL solution C (35 mL absolute ethanol + 15 mLTE) to the spin column, and centrifuge at 8000 rpm for 1 min.

6. Discard the liquid, wash once again with 500 μL solution C, and centrifuge at 12,000 rpm for 1 min.

7. Place the spin column in the air for 5-10 minutes to remove residual ethanol.

8. Put the spin column in a new Eppendorf tube, add 20-30 μL sterile water or TE (pH 8.0), and let stand at 50°C for 2 minutes.

9. Centrifuge at 12000rpm for 1min, and the solution at the bottom of the Eppendorf tube will contain the recovered DNA fragments.

5. Connection

Mix 1 times the vector pBS-SV digested fragment and 3 times the genomic DNA fragment in a microcentrifuge tube, add 1 μL 10× Ligase Buffer and 0.5 μL T ₄ DNA Ligase, make up the reaction to 10 μL, and the whole reaction Incubate overnight at 16°C. The ligation product was precipitated with ethanol to purify the ligation system and inactivate the ligase. The purified ligation product can be directly used for transformation or stored at -20°C for future use.

6. Transformation

1. Take 10 μL of the connection solution and add it to 100 μL of competent cells, and place in an ice bath for 30 minutes.

2. Heat shock treatment at 42°C for 60-90s, do not shake during this process.

3. Quickly transfer the tube to an ice bath and allow the cells to cool for 5 minutes.

4. In a sterile ultra-clean bench, add 900 μL sterile LB medium to each tube.

5. Transfer the microcentrifuge tube to a 37°C incubator and let it stand for 10 minutes.

6. Then transfer the microcentrifuge tube to a shaker at 37°C, and incubate with shaking for 50 minutes.

7. Spread an appropriate volume of bacterial liquid on an agar solid plate containing 50ug/mL ampicillin, X-Gal and IPTG. Invert the plate and incubate at a constant temperature of 37°C for 16h.

7. Resistance screening

1. Weigh the following reagents and place them in a 1L beaker.

Tryptone 10g

Yeast Extract 5g

Sodium chloride 10g

2. Add about 800mL of deionized water and stir well to dissolve.

3. Add 5N NaOH solution (about 0.2mL) dropwise to adjust the pH value to 7.0.

4. Add dehydrated ionized water to adjust the volume of the medium to 1L, and then add 15g of Agar. After high temperature and high pressure sterilization, cool to about 60°C.

5. Add 0.5mL ampicillin (100mg/mL) to 1L medium and mix evenly, divide into several portions, each portion is about 25mL.

7. Add different amounts of kanamycin in a gradient to the well-divided small portion of the medium, and spread the plate.

8. Pick the single clone containing random sequence after transformation, and draw the plate on the plate containing kanamycin.

9. Incubate at a constant temperature of 37°C for 16 hours, observe the growth, and pick well-growing single clones for identification of resistance to kanamycin at different concentrations.

10. Sequence the positive clones to obtain the gene sequence that can resist kanamycin.

Claims (9)

1. novel method of setting up vast capacity gene library based on combinatorial principle and PCR.

Step comprises:

(a) design DNA or RNA sequence at random, there is the shared joints sequence at the two ends of sequence.

(b) utilize the complementation of shared joints sequence, stochastic sequence is carried out PCR meet again in solution.

(c) add the length that the Auele Specific Primer that comprises (a) center tap sequence is controlled the reunion product in the reunion process or after the reunion process.

(d) utilize the PCR means reunion product to be increased with Auele Specific Primer.

(e) to (b) or (c) or after DNA (d) or RNA product obtain single chain molecule with asymmetric PCR, directly screen, obtain discerning mutually and specificity bonded dna molecular or RNA molecule with target substance.

(f) to (b) (c) or DNA product (d) carry out fragment and reclaim, by enzyme cut, connect, means such as conversion, screening obtains discerning mutually with target substance and specificity bonded protein or the protein of other any function is arranged.

2. method according to claim 1 is characterized in that: the DNA stochastic sequence in the step (a) partly is NNN, perhaps NNY or NNR.Wherein N is C, G, A, one of four bases of T; Y is one of C or two bases of T; R is one of A or two bases of G.The number of stochastic sequence is a 3-300 base.

3. method according to claim 1 is characterized in that: the RNA sequence in the step (a), its stochastic sequence partly are NNN, perhaps NNY or NNR, and wherein N is C, G, A, one of four bases of U; Y is one of C or two bases of U; R is one of A or two bases of G.The number of stochastic sequence is a 3-300 base.

4. method according to claim 1, it is characterized in that: the shared joints part at DNA in the step (a) or RNA stochastic sequence two ends, its 5 ' end is complementary with 3 ' end of another shared joints sequence, and 3 ' end is complementary with 5 ' end of another shared joints sequence.The number of complementary base is 6-30.

5. method according to claim 1 is characterized in that: the stochastic sequence PCR in the step (b) meets again, and the complementation owing to joint sequence between the stochastic sequence is met again, and the sequence after the reunion comprises the random fragment more than three sections and three sections.

6. method according to claim 1 is characterized in that: the stochastic sequence PCR in the step (b) meets again, and the total concn of all initial random fragments is at 5ng/ μ L-200ng/ μ L.

7. method according to claim 1 is characterized in that: to step (b) or the reunion fragment that (c) produces, carry out the amplification of sequence with the PCR means.

8. method according to claim 1, it is characterized in that: or (c) or DNA (d) or RNA product step (b), after obtaining single chain molecule with asymmetric PCR, directly carry out discerning mutually and the screening of specificity bonded, obtain discerning mutually and specificity bonded dna molecular or RNA molecule with target substance with target substance.

9. method according to claim 1, it is characterized in that: to step (b) (c) or reunion DNA product (d) carry out means such as enzyme is cut, connected, conversion, carry out discerning with specificity bonded protein mutually with target substance or the proteinic screening of other any function being arranged.

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