CN104789555A - Method of expressing regulatory element based on evolution gene by synthesizing single-chain DNA library - Google Patents

Abstract

The invention discloses a method of expressing a regulatory element based on an evolution gene by synthesizing a single-chain DNA library and belongs to the technical field of genetic engineering. The method comprises the following steps: based on a PCR technology, a single-chain mutant is obtained firstly, and then a double-chain mutant library which is rich in diversity with the single-chain mutant as a template is obtained; and after an expression vector of the mutation library is recombined, a host is transformed, and high throughput screening is carried out on evolved mutant strains. According to the method disclosed by the invention, with the adoption of the technology, transformation in vitro is successfully carried out on a promoter, and the mutant strain with very excellent property is obtained; and rapid evolution in vitro of the gene is realized, and the method is simple to operate and is particularly suitable for carrying out rapid oriented evolution for introducing rich mutation diversity in the gene.

Description

A Method for Evolving Gene Expression Regulatory Elements Based on Synthetic Single-Stranded DNA Libraries

technical field

The invention relates to a method for evolving gene expression regulation elements based on a synthetic single-stranded DNA library, and belongs to the technical field of genetic engineering.

Background technique

With the development of molecular biology technology and genetic engineering technology, thousands of natural enzyme genes have been discovered. Because enzyme-mediated biocatalysis has the advantages of environmental friendliness, high efficiency and specificity, and mild reaction conditions, it is used as a biocatalyst. The leader of catalysis is widely used in the synthesis of complex drugs, intermediates, compounds, and even the synthesis of biofuels. Enzymes are the core participants in synthetic biology and metabolic engineering. By constructing a series of metabolic synthesis pathways, microorganisms can metabolize and synthesize various complex compounds that cannot be obtained by organic synthesis, such as aromatic hydrocarbons, carbohydrates, organic acids, alcohols and other secondary metabolite. However, in most cases, limitations in the properties of natural enzymes greatly limit their use in a variety of complex conditions.

In the dynamic balance process of genetic mutation and natural selection, nature has evolved very precise and efficient enzymes, but the speed of natural selection is very slow and takes a long time. There is an urgent need for artificial evolution and modification of natural enzymes in order to obtain new enzymes with more powerful functions that can meet people's needs. Under the conditions of today's experimental technology, people have greatly increased the frequency of gene mutations through physical and chemical methods, such as ultraviolet light, radiation, and nitrite, providing the possibility for artificial rapid evolution of genes. However, the mutations caused by physical and chemical methods are often harmful mutations, and they are relatively random, which cannot meet the needs of large-scale evolution. With the maturity of molecular biology technology, it has become possible to directly introduce specific or random mutation sites into natural genes from the level of genetic manipulation. The random mutation method is to screen ideal mutants by introducing random base substitutions. For example, error-prone PCR technology is one of the most widely used gene evolution methods at present. Through the random introduction of mutation points in the in vitro polymerase chain reaction (PCR) process, high-throughput screening of genes beneficial to evolution is performed. After multiple rounds of operations, new enzymes with significant changes in enzymatic properties and functions can be obtained. Various technologies developed on this basis rely on PCR technology to introduce mutation point evolutionary genes, such as site-directed mutagenesis, gene chimerism, and random chimerism of transient templates. However, the mutation point diversity generated by the above method is small, and most of the mutants generated are harmful mutations, the workload is heavy and the efficiency is extremely low.

In 1994, a new technology of directed evolution, DNA shuffling, was quietly born. Stemmer published an article titled Rapid evolution of a protein in vitro by DNA shuffling in Nature, and proposed the DNA shuffling technology for the first time. The researchers took the β-lactamase system as the test object, used DNA shuffling, and obtained a new strain after three rounds of screening and two backcrosses. The minimum inhibitory concentration of cefotaxime was 16,000 times higher than that of the original strain, far Mutation screening results higher than error-prone PCR. Various methods based on recombination technology developed subsequently aimed at improving the degree of gene hybridization and screening efficiency, and achieved relatively good results. However, DNA shuffling technology requires high sequence homology (homology greater than 75%) and needs to obtain homologous genetic material. These two conditions greatly limit the application of DNA shuffling technology, and the mutants produced by this technology often have more frameshift mutants.

In addition, the current modification of synthetic biology and metabolic engineering to synthetic pathways basically focuses on the expression regulation of synthetic pathway genes, such as enhancing the expression of pathway genes, knocking out or inhibiting competing pathways, such as optimizing promoters or ribosome binding sites Points, optimize the codons of genes, modify the spacer regulatory regions of pathway genes, etc. In addition, it is also very necessary to modify the enzymes in the synthetic pathway, such as the rate-limiting problem of key enzymes, feedback inhibition by products or substrates. Because, if only the expression level is enhanced, it may lead to the waste of cell synthesis capacity and the burden of cell growth, but cannot fundamentally solve the problem of metabolic pathway flow balance.

The invention adopts in vitro PCR recombination technology as the basis, artificially synthesizes a single-stranded DNA library, adopts PCR technology to obtain a mutation library with extremely rich diversity, and then uses in vitro recombination technology to construct a mutation screening library. This technology overcomes the aforementioned factors such as insufficient diversity of random mutations, limitations of DNA shuffling technology homologous sequences and homologous gene materials, etc., and is easy to operate and implement, and it is easy to obtain abundant mutant libraries, laying the foundation for the rapid evolution of genes in vitro, and at the same time , combined with protein structure bioinformatics and synthetic biology, especially suitable for rapid directed evolution applications of enzyme genes and metabolic synthetic pathways.

Contents of the invention

The present invention provides a method (Rapidly Efficient Combinatorial Oligonucleotides for Direct Evolution, RECODE) based on synthetic single-stranded DNA library evolution gene expression regulatory elements, mainly including the preparation of mutant library, the mutant library includes single-stranded mutant library and double Chain mutant library. The single-chain mutant library and the double-chain mutant library can be constructed step by step or simultaneously. In order to construct a single-stranded mutant library, primers were designed for each target mutation region of the parental gene. Strand mutants: In order to construct a double-stranded mutant library, the two ends of the single-stranded mutants need to carry additional anchor sequences to facilitate the design of primers for the specific amplification of single-stranded mutants when double-stranded.

The target mutation region can be purposefully selected or randomly generated. The number of regions for simultaneous mutation on a chain is not limited in theory, and can be set according to actual needs in practice. The number of genes within 2k can be between 1-15 sites.

The same direction means that each primer is in the 3' to 5' direction, or in the 5' to 3' direction. When the parental gene is double-stranded DNA, the same primer direction can ensure that the PCR process only uses the DNA strand in a specific direction as a template, and finally obtains a single-stranded mutant.

The parental genes encode promoters, ribosome binding sites or other gene expression regulatory elements. The form of the parental gene is not limited, and may be a plasmid, genome, double-stranded DNA fragment or single-stranded cDNA.

The synchronous construction of the single-stranded mutant library and the double-stranded mutant library is to directly add primers that specifically bind to single-stranded mutants to the PCR system for constructing the single-stranded mutant library, and obtain single-stranded mutants by PCR. The double-stranded mutant is used as a template for double-stranded reaction and double-stranded mutant amplification reaction.

When the single-stranded mutant library and the double-stranded mutant library are constructed step by step, the PCR mixture containing the single-stranded mutant library is directly used as a template, and the reaction system is not changed, and primers that specifically bind to the single-stranded mutant are directly added thereto. The second round of PCR prepares full-length and complete double-stranded mutants; or uses the single-stranded mutant library recovered after column purification as a template to reconstitute the reaction system for the second round of PCR to prepare full-length and complete double-stranded mutants.

To construct a single-stranded mutant library, corresponding primers are designed for one or more target mutation regions of the parental gene, called editing primers. The editing primers contain the mutated bases that need to be introduced into the mutated region, and the 3' and 5' ends have sequences matching the template strand of appropriate length; Template strands combine in pairs. At the same time, synthesize two anchor primers in the same direction as the editing primer, one of which is an upstream anchor primer and one is a downstream anchor primer, and the upstream anchor primer contains a nucleotide sequence that matches the 3' end of the template strand , In addition, the 5' end of the upstream anchor primer also has an anchor sequence of 15-25bp; the downstream anchor primer contains a nucleotide sequence that matches the 5' end of the template chain, in addition, the downstream anchor primer There is also a 15-25bp anchor sequence at the 3' end.

For editing primers, in order to save synthesis costs, it is generally recommended that the length of editing primers be less than 59 bp, if necessary, the length of the primers can be increased, or it can be divided into two short primers. The site that needs to be mutated is generally placed in the middle of the editing primer, and can be highly degenerated as needed. If the parental code is a protein sequence, in order to avoid the occurrence of stop codons TAA, TAG and TGA, the triple codon can be degenerated as follows: VNN may reduce the probability of terminator occurrence, and editing primer synthesis is preferred for PAGE purification.

The anchor primer can also function as an editing primer, that is, while serving as an anchor primer, it can also contain a mutated base that needs to be introduced into the mutated region.

The anchor point sequence, on the one hand, can be used to design an outer primer specifically combined with a single-stranded mutant when constructing a double-stranded mutant library; on the other hand, it can also be used as a homologous recombination sequence for the mutant library Ligation to expression vectors. When linking double-stranded mutants to the vector, it is not recommended to use restriction enzyme sites for recombination vectors, because the introduction of highly degenerate mutations may cause corresponding enzyme sites to appear inside the gene.

The mutated bases to be introduced in the mutated region may be a specific base sequence or a degenerate base sequence.

Before using the editing primer and the downstream anchor primer, T4 polynucleotide kinase is used to phosphorylate the 5' terminal hydroxyl of the primer.

The 3' and 5' end sequences of the editing primers respectively retain 15-30 bp oligonucleotide sequences that completely match the template.

The reaction process of preparing a single-stranded mutant library is a simultaneous reaction of DNA extension and DNA ligation: upstream and downstream anchor primers, editing primers and template DNA are mixed at an appropriate molar ratio, DNA polymerase and DNA ligase are added, and reaction buffer . The Mg ²⁺ concentration in the reaction buffer is 1-10mM, the PCR annealing temperature is 40-66°C, the PCR extension temperature is 65-72°C, and the number of PCR reaction cycles is 1-35. The PCR products include single-stranded DNA that is not used as a template after denaturation and melting, a large number of mutated single-stranded DNA, and mutated DNA strands that are in a double-stranded state combined with the template.

In one embodiment of the present invention, when preparing a single-stranded mutant library, the number of moles of primers that bind to different positions of the template strand is the same.

To construct a double-stranded mutant library, the PCR mixture containing the single-stranded mutant library or the single-stranded mutant library recovered after column purification can be used as a template to perform a second round of PCR to prepare a full-length and complete double-stranded mutant gene product or directly add primers that specifically bind to single-stranded mutants in the reaction system for constructing single-stranded mutant libraries, and carry out double-strandization reactions and amplification reactions of double-stranded mutants (referred to as one-step for short) while obtaining single-stranded mutants by PCR. PCR). In order to construct a double-stranded mutant library, a pair of outer primers for amplifying the full-length gene of the single-stranded mutant is designed using the single-stranded mutant as a template, and the outer primers match the anchor sequences of the anchor primers respectively or the same. Only the mutated single-stranded DNA with the anchor sequence can combine with the outer primer, so the amplified product is a library of double-stranded mutants. After the prepared PCR products are purified and recovered, they are cloned using in vitro recombination technology to construct a high-throughput screening library to screen for mutants with the desired properties and characteristics.

When the PCR mixture containing the single-stranded mutant library or the single-stranded mutant library recovered after column purification is used as a template for the second round of PCR to prepare a full-length complete double-stranded mutant gene product, only a few cycles are required. A large number of double-stranded mutants can be obtained. For the single-strand mutant library, before the double-strand mutant library is prepared, the template strand can be digested with DpnI restriction endonuclease. Of course, even if DpnI restriction endonuclease is not used to degrade the template DNA, during the PCR process of preparing the double-stranded mutant library, since a pair of outer primers contain a sequence matching the anchor point sequence, the template DNA does not contain a sequence matching the anchor point sequence. sequence, therefore, template DNA does not amplify.

The one-step PCR of directly adding primers that specifically bind to single-stranded mutants to the reaction system for constructing a single-stranded mutant library, obtaining single-stranded mutants by PCR, and performing double-stranded reaction and amplification reaction of double-stranded mutants In the method, mix the upstream and downstream anchor primers, editing primers and template DNA at an appropriate molar ratio, and at the same time, add 2-5 times the amount of the anchor primer, the upstream and downstream outer primers, add DNA polymerase, DNA ligase and Reaction buffer. The Mg ²⁺ concentration in the reaction buffer is 1-10mM, the PCR annealing temperature is 40-66°C, the PCR extension temperature is 65-72°C, and the number of PCR reaction cycles is 1-35. A large number of double-stranded mutant libraries of PCR products, and a very small amount of template DNA.

During the annealing process of the one-step PCR, in order to avoid the combination of the anchor primer and the outer primer, the length of the anchor sequence in the anchor primer accounts for about one-third of the length of the anchor primer.

In the one-step PCR, the length of the upstream (downstream) anchor primer can be 60bp, of which 40bp is completely matched with the parental template, and there is also a 20bp anchor sequence; The sequences are identical or complementary.

In the one-step PCR, the addition ratio of the upstream (downstream) anchor primer and the upstream (downstream) outer primer can be 1: (2-5), and the excess upstream (downstream) outer primer can make the generated Single-stranded mutants are fully duplexed to double-stranded mutants.

In one embodiment of the present invention, the DNA polymerase used is a high-fidelity thermostable DNA polymerase, preferably a high-fidelity DNA polymerase whose 5'-3' exonuclease activity is missing, such as Phusion DNA polymerase (NEB) .

In one embodiment of the present invention, the DNA ligase used is a thermostable DNA ligase, such as Taq DNA ligase (NEB), 9N DNA ligase (NEB) and Ampligase (EPI), preferably Ampligase.

To screen mutants, mutant libraries and vectors can be integrated with in vitro assembly techniques.

The in vitro assembly technology may be fusion PCR, Gibson assembly, T4 DNA polymerase-mediated recombination technology or in vivo yeast assembly technology. The principle of Gibson assembly technology is that in the assembly reaction system, T5 exonuclease can cut from the 5' end of the double-stranded DNA fragment to produce a sticky end protruding from the 3' end. Arm sequence, two adjacent completely complementary cohesive end fragments are combined, and the resulting gap is filled by DNA polymerase and ligated by Taq DNA ligase, so as to realize the seamless assembly of recombinant fragments.

Fig. 1 can help to understand the principle of the present invention, and Fig. 1 does not represent the only embodiment of the present invention. The present invention mainly includes two rounds of PCR respectively using different primers and the process of screening the second round of PCR products. The first round of PCR involves five primers in the 5' to 3' direction shown at the top of Figure 1, among which the two primers with dotted lines are anchor primers, and the part indicated by the dotted line is the anchor sequence, marked with ★, ▲ , ◆ are editing primers designed for different target mutation regions; during the denaturation stage of the PCR process, the parental DNA melts to obtain two single-stranded DNAs, since the five primers can only combine with the DNA strand in the 3' to 5' direction Therefore, only the parental DNA strand in the 3' to 5' direction can be used as the template strand during the entire PCR process. Single-stranded DNA used as a template, and a small amount of mutated DNA strand in a double-stranded state bound to the template. Use the mixed product of the first round of PCR as a template, or recover the single-stranded mutant as a template by using DNA solution column purification method to reduce other influencing factors. Correspondingly, the second round of PCR may not change the reaction system, directly add the upstream and downstream outer end primers to the first round of PCR mixed product, or reconfigure the PCR reaction system. The second round of PCR involves a pair of outer primers that match or are identical to the anchor sequence of the anchor primer in the first round of PCR, so that the outer primers only bind to single-stranded DNA with the anchor sequence Amplification reaction, while the parental DNA is not amplified due to the lack of a sequence that matches the anchor sequence. Finally, the ideal mutant is obtained by screening the abundant double-stranded mutant library.

Fig. 4 is a simplified one-step PCR obtained on the basis of Fig. 1, which can directly prepare a double-stranded mutant library. One-step PCR is to add the upper and lower outer end primers in the first round of PCR reaction system, so that during the reaction process, the newly generated single-stranded mutants are combined with the upstream or downstream outer end primers and amplified to form double-stranded mutants. chain mutants, and the parental chain will not be amplified because it cannot bind to the outer primer. During this process, the mutants formed grow exponentially, and more mutants are produced than in the two-round PCR reaction system. At the same time, it is more convenient and time-consuming in operation.

Based on the in vitro PCR recombination technology, the present invention artificially synthesizes a single-stranded mutant library. By designing editing primers for multiple target regions, a mutant library with extremely rich diversity is obtained after PCR, and the in vitro recombination technology is used after double-stranding Construction of mutant screening libraries. The present invention overcomes the disadvantages of lack of diversity in existing random mutations, and DNA shuffling technology is limited by homologous sequences and homologous gene materials, and is easy to operate and implement, and is easy to obtain abundant mutant libraries, laying the foundation for the rapid evolution of genes in vitro. Objects for rapid and efficient evolution include multiple DNA sequences such as promoters, ribosome binding sites, and non-transcribed translational regulatory regions.

Description of drawings

Figure 1 is a schematic diagram of the method for the rapid combinatorial evolution of enzymes and metabolic pathways by synthesizing a single-stranded DNA library; the dotted lines at both ends are anchor sequences.

Figure 2 is a schematic diagram of the fluorescence intensity of each mutant unit cell in the mutant library, and the arrow indicates the unit fluorescence intensity value of the wild-type promoter.

Fig. 3 is a schematic diagram showing the sequence alignment of rpoS mutant promoters with different strengths.

Fig. 4 is a schematic diagram of a method for combining and evolving DNA using a one-step PCR reaction system in RECODE technology; the dotted lines at both ends are anchor point sequences.

Detailed ways

Example 1 Application of one-step PCR to transform the constitutive promoter of E. coli rpoS gene in vitro

Taking the constitutive promoter of the rpoS gene of Escherichia coli as an example, the nucleotide sequence is shown in SEQ ID NO.1, and the four internal spacer sequences of -10 and -35 intervals were combined and mutated at the same time to construct a mutant library. Four editing primers were designed according to the sequence of the promoter segment, editing primers (Jps/rpoSp-F, Jps/rpoSp4-F, Jps/rpoSp3-F, Jps/rpoSp21-F), anchor primers (Jps/rpoSp-HM -F, Jps/rpoSp-HM-R) outer primers (rpoSp-F, rpoSp-R) and vector preparation primers are as follows:

Jps/rpoSp-F:TTCCACCGTTGCTGTTGCGTNNNNNNNNNNNNNNNNTATTCTGAGTCTTCGGGTGAAC

Jps/rpoSp4-F:CATAACGACACAATGCTGGTNNNNNNNNNNNNNAAGTTAAGGCGGGGCAAAAAATAGC

Jps/rpoSp3-F:TAGCACCGGAACCAGTTCAANNNNNNNNNNNNNNNNAATTCGTTACAAGGGGAAATCCG

Jps/rpoSp21-F:GCAGCGATAAATCGGCGGAACNNNNNNNNNNNNNNNNNTGNTCCGTCAAGGGATCACGGG

Jps/rpoSp-HM-F:CACTATAGGGCGAATTGGAGCTCCATACGCGCTGAACGTTGGTCAG

Jps/rpoSp-HM-R:TCAAGGGATCACGGGTAGGAGCCACCGATCCATGGGTAAGGGAGAAGAAC

rpoSp-F:CACTATAGGGCGAATTGGAGCT

rpoSp-R:GTTCTTCTCCCCTTACCCATGGATC

PBBR2-gfp-F:CCATGGGTAAGGGAGAAGAAC

PBBR2-gfp-R:CCGGAATTCTTATTTGTATAGTTCATCCATG

The two anchor primers Jps/rpoSp-HM-F and Jps/rpoSp-HM-R are in the same direction as the editing primers, and both are paired with the same single-stranded template. The anchor primers have an anchor sequence of more than 20 bp, and the anchor The point sequence is consistent with the gap position sequence of the vector pBBRMCS2-gfp, which is used for homologous recombination cloning. The cloning vector was amplified with PBBR2-gfp-F and PBBR2-gfp-R primers. The insertion position of the mutant promoter was located upstream of the green fluorescent protein gfp, which was used to drive the expression of GFP protein. The intensity of the promoter was detected by the fluorescence intensity of the reporter gene GFP. weak. First, the editing primers Jps/rpoSp-F, Jps/rpoSp4-F, Jps/rpoSp3-F, Jps/rpoSp21-F and the downstream anchor primer Jps/rpoSp-HM-R were mixed in an equimolar ratio, using T4 polynucleoside The phosphorylation reaction of acid kinase was carried out, and the reaction system was operated according to the product instructions, and the reaction was carried out at 37°C for 30 minutes, and at 70°C for 10 minutes to inactivate the enzyme.

Preparation of single-stranded mutant libraries is a simultaneous reaction of PCR cycles for DNA extension and ligation. In a 25 μl reaction system: 2.5 μl 10x reaction buffer, upstream anchor primer Jps/rpoSp-HM-F, phosphorylated downstream anchor primer and phosphorylated editing primer (calculated according to the phosphorylation system, each editing primer 1pmol), the amount of DNA template added is about 1-5ng (the ratio of primer to primer is 1:100), Phusion DNA polymerase (NEB) 0.5μl, Ampligase (EPI) ligase 1μl are added. After mixing, run according to the following PCR program: 94°C for 2min, [94°C for 30s, 50°C for 1min, 66°C for 5min] x25, 72°C for 5min, 4°C hold on. The first-round PCR product is a single-stranded mutant library, which can be used for the next step immediately or stored at -20°C.

Preparation of double-stranded mutant library: The second round of PCR is used to prepare the full-length complete gene. In a 50 μl reaction system, 25 μl 2x premixed DNA polymerase (Superpfu mix, Hangzhou Baosai Biotechnology Co., Ltd.), and the two ends of the full-length gene 1 μl (10 μM) of each of the outer primers rpoSp-F and rpoSp-R, and 5 μl of the single-strand reaction solution prepared in the above steps as a template, amplified according to the following PCR program: 94°C for 3 minutes, [94°C for 30 seconds, 55°C for 30 minutes, 68°C 1min] x32, 72°C 5min, 4°C hold on. PCR products were recovered by 1% agarose gel electrophoresis. The PCR product was recovered using the gel recovery kit according to the method recommended in the manual.

The assembly of the mutant library and the vector adopts the Gibson assembly technology. The Gibson assembly technology refers to the literature Daniel G Gibson.et al.2009.NATURE METHODS VOL.6NO.5 343-345. 5×reaction buffer configuration: 25% PEG-8000, 500mM Tris-HCl pH 7.5, 50 mM MgCl ₂ , 50 mM DTT, 1 mM each of dNTPs and 5 mM NAD. The 20 μl reaction system contained 8 μl 5× reaction buffer, 0.8 μl T5exonuclease (0.2U/μl Epicentre), 4 μl TaqDNA ligase (40U/μl New England Biolabs (NEB)) and 0.5 μl Phusion DNA polymerase (2U/μl NEB). At the same time, 50 ng of the linearized vector was added, and the amount of the mutant library fragment and the vector was in an equal molar ratio. After mixing the reaction system, react at 50°C for 60 minutes. All the reaction liquids were transformed into E. coli competent cells, and operated according to the normal transformation steps. After incubating at 37°C for 1 hour, all were coated with LB plates containing 50 μg/ml kanapenicillin, and incubated overnight at 37°C.

High-throughput screening was performed on the grown clones, cultured in a 96-well plate, and 1000 single clones were randomly selected and inoculated in LB medium of a 96-well plate, added with 50 μg/ml kanapenicillin, and cultured for 24 hours at 37 hours. The 96-well plate culture solution was centrifuged at 3500rpm for 5 minutes, washed repeatedly with sterile water for 3 times to remove the influence of the medium, added an appropriate amount of sterile water to resuspend, and used a microplate reader to scan the fluorescence intensity and cell OD ₆₀₀ value . The strength of the mutant promoter is represented according to the fluorescence intensity of the unit thalline, and the result is shown in Figure 2, the intensity variation range of the promoter in the mutant library is between 6%-460%, thus illustrating that the method of the present invention is effective Efficient combinatorial evolution of promoters in vitro has obvious effects. At the same time, the analysis of the sequencing results is shown in Figure 3. The mutant promoters of different strengths generated have a significant change in sequence mutation diversity. In the interval region between the four promoters, as many as 10-20 mutant bases were introduced. And the mutated bases introduced at each site are different.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. to evolve the method for gene expression regulation element, it is characterized in that, mainly comprise and prepare strand mutant library, prepare double chain mutation body library, screening mutant, strand mutant library and double chain mutation body library step by step or build simultaneously; For building strand mutant library, primer is designed respectively in each targeted mutagenesis region for parental gene, each primer direction is identical, and by adding DNA ligase to connect the nucleotide fragments of respectively undergoing mutation in PCR system, obtains complete strand mutant; For building double chain mutation body library, extra anchor point sequence need be brought in the two ends of strand mutant, is convenient to when duplexed, is designed for the primer of specific amplification strand mutant.

2. method according to claim 1, is characterized in that, what described parental gene was encoded is promotor, ribosome bind site or other gene expression regulation elements.

3. method according to claim 1, is characterized in that, the existence form of described parental gene is unrestricted, can be plasmid, genome, double chain DNA fragment or strand cDNA.

4. according to the arbitrary described method of claim 1-3, it is characterized in that, for building strand mutant library, for one or more targeted mutagenesis regions of parental gene, designing corresponding primer respectively, being called editor's primer; Described editor's primer contains mutating alkali yl that needs introduce to Sudden change region and 3 ' and 5 ' end has the sequence of mating with template strand of suitable length respectively; When relating to multiple Sudden change region, each editor's primer is equidirectional, all matches with same template strand and combines; Design two and the equidirectional anchor point primer of editor's primer in addition, wherein one is upstream anchor point primer, article one, be downstream anchor point primer, described upstream anchor point primer contains one section holds with template strand 3 ' nucleotide sequence mated, in addition, 5 ' end of this upstream anchor point primer also has the anchor point sequence of 15-25bp; Described downstream anchor point primer contains one section holds with template strand 5 ' nucleotide sequence mated, and in addition, 3 ' end of this downstream anchor point primer also has the anchor point sequence of 15-25bp.

5. method according to claim 4, is characterized in that, needs the site of carrying out suddenling change to be placed in the middle part of editor's primer, can carry out height degeneracy as required.

6. method according to claim 4, is characterized in that, described anchor point primer, also has the function of editor's primer concurrently, namely while as anchor point primer, also containing the mutating alkali yl needing to introduce to Sudden change region.

7. according to the arbitrary described method of claim 1-3, it is characterized in that, described strand mutant library and double chain mutation body library build simultaneously, it is the primer adding specific combination strand mutant in the PCR system directly to structure strand mutant library, limit PCR obtains strand mutant, while with strand mutant for template carries out the amplified reaction of duplexed reaction and double chain mutation body; When described strand mutant library and double chain mutation body library substep build, directly using the PCR mix products containing strand mutant library as template, do not change reaction system, carry out second directly to the primer wherein adding specific combination strand mutant to take turns PCR and prepare the complete double chain mutation body of total length, or using the strand mutant library after reclaiming through column purification as template, again prepare reaction system and carry out second and take turns PCR and prepare the complete double chain mutation body of total length; For building double chain mutation body library, using strand mutant as template, design a pair and match or the identical external primer for specific amplified strand mutant with anchor point sequence, the single stranded DNA of only undergoing mutation can be combined with external primer.

8. according to the arbitrary described method of claim 1-3, it is characterized in that, after prepared by described strand mutant library, before the preparation carrying out double chain mutation body library, adopt DpnI digestion with restriction enzyme template strand.

9. according to the arbitrary described method of claim 1-3, it is characterized in that, during screening mutant, double-mutant is connected with carrier by application assembled in vitro technology.

10. method according to claim 9, is characterized in that, described assembled in vitro technology is yeast package technique in fusion DNA vaccine, Gibson assembling, the polymerase-mediated recombinant technology of T4DNA or body.