CN102634535B - Construction method of zinc finger protein inserting vector based random zinc finger protein library - Google Patents

Abstract

The invention discloses a method for constructing a random zinc finger protein library, including the construction of a zinc finger protein expression insertion vector pBD-P-Gal11p-ZFR, the construction of a random zinc finger protein sequence, and the random zinc finger protein expression vector pBD-P- Gal11p-ZFS was constructed, and a high-capacity random zinc finger protein library was obtained after it was transformed and expressed. This high-capacity random zinc finger protein library lays the foundation for the screening of zinc finger proteins.

Description

A method for constructing random zinc finger protein library based on zinc finger protein insertion vector

technical field

The invention belongs to the technical field of zinc finger protein libraries, and relates to a method for constructing a random zinc finger protein library based on zinc finger protein insertion vectors.

Background technique

Zinc finger proteins are widely distributed in animals, plants and microorganisms, among which nearly 1% of the human genome encodes proteins with zinc finger structure. The common feature of zinc ^finger proteins is that the protein folds itself into Finger-like polypeptide structure. According to the number and position of Cys(C) and His(H) residues, zinc finger proteins can be divided into CCHH, CCCH-CCCC, CCHC, CCCH and other subclasses.

The C ₂ H ₂ zinc finger domain was discovered in Xenopus laevis oocyte transcription factor TFIIIA in 1983, and it is the most widely distributed protein in eukaryotic genomes so far. This zinc finger protein consists of about 30 amino acid residues in a ββα structure, and each zinc finger binds three consecutive nucleotide sequences. Its structure is relatively fixed and its affinity is strong, so it is very suitable for designing artificial DNA binding proteins.

There are currently 888 zinc fingers in the ZiFDB (http://bindr.gdcb.iastate.edu:8080/ZiFDB/) database, mainly including four categories:

SangamoBioSciences: They designed a triplet zinc finger protein, where each zinc finger recognizes the GNN nucleotide sequence.

Barbas laboratory: They used the modular assembly method to design a large number of zinc finger proteins that can recognize different triplet nucleotide sequences.

Toolgen: They screened zinc finger proteins from zinc finger proteins encoded in the human genome.

Joung laboratory: They used the OPEN method to create a large number of triplet zinc finger proteins, which recognize a 9bp target sequence.

The methods for obtaining artificial conjoined zinc fingers can be divided into two categories: screening methods and modular assembly methods. The advantage of the module assembly method is that it is simple and fast: simply connect each zinc finger module to identify the target sequence. The disadvantage is that the designed conjoined zinc fingers have low or no affinity. With the in-depth study of the mechanism of zinc finger binding to DNA, this method will become an efficient method; the screening method is more reliable, but it is time-consuming and requires higher molecular experimental skills than the module assembly method.

Description of the invention

The problem to be solved by the present invention is to provide a method for constructing a random zinc finger protein library based on zinc finger protein insertion vectors. By constructing and inserting degenerate random zinc finger protein expression sequences into expression vectors for transformation, high The capacity of the random zinc finger protein library lays the foundation for the screening of zinc finger proteins.

The present invention is realized through the following technical solutions:

A zinc finger protein insertion vector includes a promoter and an antibiotic screening gene, contains a Gal11p gene sequence downstream of the promoter, and contains a zinc finger protein sequence insertion site downstream of the Gal11p gene sequence.

The promoter is a Lac-UV5 promoter; the nucleotide sequence of the zinc finger protein sequence insertion site is shown in SEQ.ID.NO.3.

The zinc finger protein insertion vector is pBD-P-Gal11p-ZFR, the lac-UV5 promoter is cloned into the vector pBD through the restriction site, and then the Gal11p gene sequence is cloned downstream of the lac-UV5 promoter, and then The insertion site ZFR of the zinc finger protein sequence was cloned downstream of the Gal11p gene sequence.

The nucleotide sequence of the Lac-UV5 promoter shown is shown in SEQ.ID.NO.1; the nucleotide sequence of the Gal11p gene sequence is shown in SEQ.ID.NO.2; the zinc finger protein sequence insertion site The nucleotide sequence is shown in SEQ.ID.NO.3.

A random zinc finger protein sequence, the nucleotide sequence of which is shown in SEQ.ID.NO.4.

A random zinc finger protein expression vector, which is to insert the random zinc finger protein sequence shown in SEQ.ID.NO.4 with cohesive ends into the insertion site of the zinc finger protein sequence of pBD-P-Gal11p-ZFR carrier ZFR;

The pBD-P-Gal11p-ZFR vector is to clone the Lac-UV5 promoter into the vector pBD through the restriction site, then clone the Gal11p gene sequence to the downstream of the Lac-UV5 promoter, and then clone the zinc finger protein sequence The insertion site ZFR was cloned downstream of the Gal11p gene sequence.

A method for constructing a random zinc finger protein library, comprising the following steps:

1) Clone the Lac-UV5 promoter element, the Gal11p gene sequence and the insertion site ZFR of the zinc finger protein sequence shown in SEQ. Cloned into the pBD vector to construct the pBD-P-Gal11p-ZFR vector;

2) Design random primers according to the -1, 1, 2, 3, 5, and 6 positions of the zinc finger protein, and obtain the random zinc finger protein sequence shown in SEQ.ID.NO.4 by overlapping PCR;

3) Insert the random zinc finger protein sequence shown in SEQ.ID.NO.4 into the insertion site ZFR of the zinc finger protein sequence of the pBD-P-Gal11p-ZFR vector to obtain pBD-P-Gal11p- ZFS expression vector;

4) Using E.coli DH5a as the host bacterium, transforming the host bacterium with the pBD-P-Gal11p-ZFS expression vector, collecting all the single clones after chloramphenicol resistance screening, extracting the plasmids, mixing all the obtained plasmids, Obtain a library of random zinc finger proteins.

The enzyme cutting of the cohesive end is to use pfu DNA polymerase endonuclease activity to carry out enzyme cutting on the random zinc finger protein sequence.

The random zinc finger protein sequence is inserted into the pBD-P-Gal11p-ZFR vector through the BbsI restriction site.

The transfection of the pBD-P-Gal11p-ZFS expression vector adopts the method of electric transformation. Electroporation conditions, electroporation instrument parameters: voltage 1800V, duration 5ms; electric shock cup specifications: Gap (mm) 2.0, Minimum Volume 40μl, Maximum Volume 400μl.

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

At present, there are few reports on the construction method of zinc finger protein library. The most commonly used zinc finger protein library is to purchase a single-finger zinc finger library from Joung Lab, and then construct the required zinc finger library by overlapping PCR. However, there are 74 single-finger zinc-finger libraries, and each library is sold for 65$ separately, and it costs 4440$ to purchase all single-finger zinc-finger libraries (http://www.addgene.org/zfc/browse/). The procedure is cumbersome due to the need to deliver plasmids from abroad.

The present invention designs merger primers based on the structural characteristics of zinc finger proteins, and constructs a random zinc finger library by overlapping PCR, which has the following advantages:

1) The construction method is simple, and it can be completed using conventional methods of molecular biology, and basically all molecular biology laboratories have this condition. Only two instruments, a PCR machine and an electroporation machine, are needed, and it can be completed by PCR, enzyme cutting, ligation, electrotransformation and plasmid extraction.

2) The cost is low. The main costs of this method are: merger primer synthesis, restriction endonuclease Bbs I, electric shock cup, dTTP and some common laboratory reagents and equipment costs.

3) The cycle is short. The zinc finger protein is inserted into the pBD-P-Gal11p-ZFR vector to construct. It only needs to insert 3 DNA fragments into the backbone vector, which takes about two weeks. The construction of the random zinc finger library only needs to insert the random zinc finger protein sequence into the pBD-P-Gal11p-ZFR vector, then electrotransform the host bacteria, and extract the plasmid, which can be completed within 3 days.

4) The library has a large capacity. This random zinc finger protein library contains all possible zinc finger proteins that can recognize 9bp nucleotide sequences, and contains about 1.41×10 ²² different zinc finger proteins.

Description of drawings

Fig. 1 is the result diagram of single enzyme digestion identification of pBD-p vector;

Fig. 2 is the result figure of PCR identification of pBD-p-gal11p carrier;

Fig. 3 is the result figure of PCR identification of pBD-P-Gal11P-ZFR carrier;

Figure 4 is a plasmid map of the pBD-p-gal11p-ZFR vector;

Figure 5 is a schematic diagram of the construction of a random zinc finger protein library;

Figure 6 is a diagram of the amplification results of the ZFF123 fragment;

Figure 7 is a plasmid map of the pBD-p-gal11p-ZFS vector;

Figure 8 shows the expression of pBD-p-gal11p-ZFS vector in BL21.

Detailed ways

The invention provides a method for constructing a random zinc finger protein library, including the construction of zinc finger protein expression insert carrying pBD-P-Gal11p-ZFR, the construction of random zinc finger protein sequence, and the random zinc finger protein expression vector pBD-P-Gal11p -ZFS, after transforming and expressing it to obtain a high-capacity library of random zinc finger proteins. The present invention will be described in detail below in conjunction with specific embodiments, which are only explanations of the present invention rather than limitations.

First provide the source or preparation of the following culture medium/carrier:

The pBD vector, pTRG-gal11P vector, and pBD-LGF2 vector were purchased from TaKaRa Company. Escherichia coli DH5α was purchased from Tiangen Company.

SOB medium modified formula (1L system): tryptone 20g; yeast extract 5g; NaCl 0.5g; KCl 10ml (250uM).

SOC medium modified formula (1L system): tryptone 20g; yeast extract 5g; NaCl 0.5g; KCl 10ml (250uM)); glucose 1.8ml (20%).

Preparation of chloramphenicol (34mg/ml): 0.34g was dissolved in 10ml of ethanol.

IPTG (100mg/ml) preparation: 1.2g was dissolved in 50ml ddH ₂ O, filtered through a 0.22um membrane filter.

1. Construction of random zinc finger insertion vector pBD-P-Gal11P-ZFR

1.1 Construction of pBD-P vector

Design primers Plac-F, Plac-R to clone the lac-UV5 promoter from the pBD vector:

Plac-F: gcttatc atc gat aagctaa ttctcactca tta;

Plac-R: gtta gcggcc gc tttgaaga cctcatacgc tgtttcctgt;

Wherein, the underlined part is the enzyme cutting site;

Amplification system of Lac-uv5 promoter: pBD vector 1ul; primer Plac-F 2ul; Plac-R 2ul; pfuDNA polymerase 1ul; pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 29ul.

Amplification conditions of the Lac-uv5 promoter: 95°C for 3min; 95°C for 30s, 58°C for 45s, 72°C for 45s, 30cycle; 72°C for 10min.

Then use Cla I and Not I to double-enzyme digest the pBD vector (remove the λCI ORF on the pBD vector) and the PCR-amplified Lac-UV5 promoter, recover the digested fragments, and ligate them overnight at 4°C with T4 DNA ligase; To transform DH5α, the smear solution was cultured upside down at 37°C on LB solid medium containing chloramphenicol resistance (34ug/ml). Single clones were picked, identified by colony PCR and sequenced, and the pBD-P vector was obtained after correct identification. The enzyme digestion and identification results of the pBD-P vector are shown in Figure 1, where M is marker (100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp); lane 4 is the pBD vector, and lanes 1, 2 and 3 are The pBD vector was single-digested (the pBD-P vector inserted into the Lac-UV5 promoter contains a Bbs I restriction site, while the backbone vector pBD does not contain a Bbs I restriction site), and the fragment size is 2511bp. The nucleotide sequence of the lac-uv5 promoter is shown in SEQ.ID.NO.1.

1.2 Construction of pBD-P-Gal11p vector

Design primers Gal11-F and Gal11-R that introduce restriction sites, and use PTRG-Gal11p as a template to amplify the Gal11P fragment:

Gal11-F: gcc gaagac a ttatgcctca acagcagcaa;

Gal11-R: gac gcggccg c caaagcttg gatttttctc a;

Wherein, the underlined part is the enzyme cutting site;

Gal11p amplification system: pTRG vector 1ul; primer Gal11-F 2ul; Ga111-R2ul; Pfu DNA polymerase 1ul; Pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 29ul.

Amplification conditions of Gal11p: 95°C for 3min; 95°C for 30s, 58°C for 45s, 72°C for 45s, 35cycle; 72°C for 10min.

Then, pBD-P and the amplified Gal11p fragment were digested with Bbs I and Not I respectively, and the digested fragments were recovered and then ligated with DNA ligase overnight at 4°C; (34ug/ml) solid culture medium at 37 ° C inverted culture. Pick a single clone, perform colony PCR and sequence identification, and obtain the pBD-P-Gal11P vector after correct identification. The PCR identification results of the pBD-P-Gal11p vector are shown in Figure 2, where M is a marker (100, 200, 300, 400, 500, 600), and lanes 1, 2, 3, 4, 5, and 7 are constructed correctly clone, and its PCR identification band size was 296bp. The nucleotide sequence of Ga111p is shown in SEQ.ID.NO.2.

1.3 Construction of pBD-P-Gal11P-ZFR vector

Primers ZFR1 and ZFR2 were designed to introduce restriction sites, and the zinc finger protein insertion sequence ZFR was obtained by overlapping PCR method:

ZFR1: cgatgcggcc gctgactaca aagattctag acccgg ggag gtcttctaag tgataa ;

ZFR2: gcatggatcc ttactactgt gcatgtcttc ttac ttatca cttagaagac ctcc ;

Wherein, the underlined part is the bridge sequence of the overlapping primers at both ends.

The zinc finger protein insertion sequence ZFR synthesized by overlapping PCR is as follows (ie, shown in SEQ.ID.NO.3):

CGAT TGACTACAAAGATTCTAGACCCGGGGAG GTCTT C TAAGTGATAAGTAA GAAGAC ATGCACAGTAGTAA ATGC

Among them, the dotted line part is the restriction site Not I and BamH I, and the solid line part is the restriction site Bbs I. The recognition site and cutting site of the Bbs I restriction endonuclease are different, and the Bbs I restriction endonuclease site can be removed after digestion through rational design to eliminate the influence of the DNA sequence of the restriction endonuclease.

ZFR amplification system: ZFR1 2ul; ZFR2 2ul; pfu DNA polymerase 1ul; pfuDNA polymerase buffer 5ul; dNTP 2ul; ddH ₂ Oul37ul.

Amplification conditions of ZFR: 94°C for 3min; 94°C for 30s, 58°C for 30s, 72°C for 45s, 25cycle; 72°C for 10min.

Then, pBD-P-Gal11P and the amplified ZFR fragment were digested with Not I and BamH I respectively, and the digested fragments were recovered and then ligated with DNA ligase overnight at 4°C; Resistant (34ug/ml) solid medium was cultured upside down at 37°C. Pick a single clone, perform colony PCR and sequence identification, and obtain the pBD-P-Gal11P-ZFR vector after correct identification.

The colony PCR identification results of the pBD-P-Gal11P-ZFR vector are shown in Figure 3, wherein M is Trans2k DNA marker (from top to bottom the size is 2000, 1000, 750, 500, 250, 100bp) 4, 6, 7, For positive clones, 1, 2, 3, 5 negative clones. According to the designed primers, the target fragment size is 753bp.

1.4 The plasmid map of the constructed pBD-P-Gal11P-ZFR vector is shown in Figure 4, where the sequence of the three inserted elements is Lac-UV5-Gal11p-ZFR;

Lac-UV5 is a medium-strength promoter responsible for the transcription and translation of Gal11p and zinc finger sequences.

Gal11p can interact with Gal4 and can regulate the transcription of promoters. It has been widely used in protein-to-protein research, such as bacterial two-hybrid systems and yeast two-hybrid systems. In this way, the constructed carrier and zinc finger protein library can be screened by the two-hybrid system.

ZFR is the insertion site of the zinc finger protein sequence, including two Bbs I restriction sites, which can be produced at the cohesive end of the zinc finger protein sequence pair after restriction, and can remove the Bbs I restriction site contained in the vector to Eliminate the influence of enzyme cleavage sites on zinc finger protein sequences.

2. Construction of random zinc finger protein sequences

The C ₂ H ₂ zinc finger domain was discovered in Xenopus laevis oocyte transcription factor TFIIIA in 1983, and it is the most widely distributed protein in eukaryotic genomes so far. This zinc finger protein consists of about 30 amino acid residues in a ββα structure, and each zinc finger binds three consecutive nucleotide sequences. Its structure is relatively fixed and its affinity is strong, so it is very suitable for designing artificial DNA binding proteins. The present invention designs a three-finger random zinc finger protein library, which recognizes 9bp nucleotide sequences.

Zinc finger protein has a conserved sequence, and its nucleic acid-binding sites are -1, 1, 2, 3, 5, and 6, that is, these six sites are variable amino acids. According to the structural characteristics and codon degeneracy of the above-mentioned zinc finger protein sequences, primers were designed, and random zinc finger protein sequences were obtained by overlapping PCR (the construction process is shown in Figure 5).

For random zinc finger protein sequences that recognize all sequences of 9bp nucleotides, the designed random primers are:

ZFF11: gagcgcccct tccagtgtcg catttgcatg cggaactttt cgvnsvnmvn vnwcttvnwvnmcatacccc gtactcatac;

ZFF12: cgcatacaga tccgacactg aaacggtttt tcaccggtat gagtacgggt atg;

ZFF21: gtgtcggatc tgtatgcgaa atttctccvn kvnmvnsvnm ttgvnwvnkc atctacgtacgcacacc;

ZFF22: tcggcattgg aatggcttct cgccggtgtg cgtacgtaga tg

ZFF31: gccattccaa tgccgaatat gcatgcgcaa cttcagt

ZFF32: ccctcaggtg ggtttttagg tgknbmnbca gsnbmnbknb mnbactgaag tgcgcatg;

ZFA1: ggggagcgcc ccttccagtg tcgc;

ZFA2: gtgcagagga tcccctcagg tgggttttta ggtg;

Among them, N represents A, T, C, G; V represents G, A, C; S represents G, C; M represents A, C; W represents A, T; K represents G, T;

The procedure for amplification of random zinc finger protein sequences is:

ZFF11 and ZFF12 amplification ZFF1, the amplification system (50ul) is: ZFF11 1ul (50uM); ZFF121ul (50uM); pfu DNA polymerase 1ul; pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 41ul;

ZFF21 and ZFF22 amplification ZFF2, the amplification system (50ul) is: ZFF211ul (50uM); ZFF221ul (50uM); Pfu DNA polymerase 1ul; pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 41ul;

ZFF31 and ZFF32 amplification ZFF3, the amplification system (50ul) is: ZFF31 1ul (50uM);

ZFF321ul(50uM); pfu DNA polymerase 1ul; pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 41ul;

The amplification conditions are: 94°C for 3 minutes; 94°C for 30s, 50°C for 30s, 72°C for 30s, 6cycles; 94°C for 30s, 56°C for 30s, 72°C for 30s, 25cycles; 72°C for 10min.

After the above amplification is completed, the amplified ZFF1, ZFF2, and ZFF3 fragments are respectively recovered, and their sequences are as follows:

ZFF-1: gagcgcccct tccagtgtcg catttgcatg cggaactttt cgvnsvnmvn mvnwcttvnwvnmcatacccc gtactcatac cggtgaaaaa ccgtttca gt gtcggatctg tatgcg

ZFF-2: gtgtcggatc tgtatgcg aa atttctccvn kvnmvnsvnm ttgvnwvnkc atctacgtacgcacaccggc gagaagccat tccaatgccg a

ZFF-3: gccattccaa tgccga atat gcatgcgcaa cttcagtvnk vnmvnkvnsc tgvnkvnmcacctaaaaacc cacctgaggg

The underlined part is the overlapping part of the three zinc finger protein sequences, and the random combination of the three sequences can be realized by overlapping PCR. Then the following procedure bridges the amplification:

ZFF1+ZFF2 amplification system amplifies ZFF12: ZFF1 1.5ul; ZFF2 1.5ul; pfu DNA polymerase 1ul; pfuDNA polymerase buffer 2ul; dNTP 1ul; ddH ₂ O 13ul.

ZFF2+ZFF3 bridge amplification system amplifies ZFF23: ZFF2 1.5ul; ZFF3 1.5ul; pfuDNA polymerase 1ul; pfu DNA polymerase buffer 2ul; dNTP 1ul; ddH ₂ O13ul

The amplification conditions are: 94°C for 3min; 94°C for 30s, 58°C for 30s, 72°C for 30s, 15cycle; 72°C for 10min.

After the amplification is completed, the amplified ZFF12 and ZFF23 fragments are recovered separately, and then the following bridge amplification is performed:

Amplification system: ZFF12 3ul; ZFF23 3ul; Pfu DNA polymerase 1ul; Pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 37ul;

Amplification conditions: 94°C for 3min; 94°C for 30s, 57°C for 30s, 72°C for 30s, 15cycle; 72°C for 10min.

After the above amplification was completed, the amplified ZFF123 fragment was recovered, and the electrophoresis detection result of the amplification result was shown in Figure 6, wherein, M was a 50bp marker, and 1 was a ZFF123 amplified fragment with a size of 283bp.

Using the ZFF123 fragment as a template, use phosphorylated primers (phosphorylated to improve subsequent connection efficiency) ZFA1 and ZFA2 to amplify ZFF123A. The amplification system is:

ZFF1233ul; ZFA1primer 1ul(50uM); ZFA2primer 1ul(50uM); pfuDNA polymerase 1ul; pfu DNA polymerase buffer 5ul; dNTP 1ul; ddH ₂ O 38ul;

The amplification conditions are: 94°C for 3min; 94°C for 30s, 58°C for 45s, 72°C for 45s, 35cycle; 72°C for 10min. The final amplified ZFF123A fragment is shown in Figure 6, where M is a 50bp marker, and 1 is the F123 amplified fragment with a size of 286bp.

The amplified ZFF 123A fragment is recovered, and its sequence is shown in SEQ.ID.NO.4.

Then through the 3'-5' exonuclease activity of pfu DNApolymerase to obtain the zinc finger protein sequence ZFS with sticky ends. Its sticky ends are as follows:

GGGG----ZFS----

----ZFS----CGTG

The enzyme digestion system: ZFF12 310ul; dTTP 3ul; pfu DNA polymerase 1ul; pfu DNA polymerase Buffer 3ul; ddH ₂ O 13ul; Thus, the zinc finger protein sequence ZFS with cohesive ends was obtained.

3. Construction of expression vector pBD-P-Gal11p-ZFS and establishment of random zinc finger protein library

After the pBD-P-Gal11p-ZFR vector was digested with BbsI (37° C. for 6 h), the digested fragment DNA was recovered. The zinc finger protein sequence ZFS with cohesive ends recovered by enzyme digestion was ligated to it, and T4 ligase was ligated overnight at 4°C; the random zinc finger expression vector pBD-P-Gal11p-ZFS was obtained;

The plasmid map of the random zinc finger expression vector pBD-P-Gal11p-ZFS is shown in Figure 7, where the inserted ZFS is located between the two BbsI restriction sites of the zinc finger protein insertion site ZFR. Transfer the zinc finger expression vector pBD-P-Gal11p-ZFS into BL21 strain to induce protein expression, culture the bacteria at 37°C to a concentration of about 0.6, add 50uM IPTG and induce at 27°C for 12 hours, collect 1ml of bacteria (12000rpm 1min), wash with PBS Add 30ul lysate [Popculture reagent (Novagen, cat.no.71092) according to 10:1 and add 4units/ml lysozyme] for 30min on ice, add loading buffer [100Mm TrisHCL (ph=6.8 ); 4% (m/v) SDS; 2% (v/v) bromophenol blue; 20% glycerol; 2% β-mercaptoethanol] 95 ° C water bath for 10 min, 10% polyacrylamide gel electrophoresis showed that ZFS can be expressed normally As shown in Figure 8, where M is Blue Plus II Protein Marker (100kDa, 62kDa, 40kDa, 30kDa, 24kDa, 14kDa from top to bottom), 1 and 2 are empty strains, 3, 4, 5, and 6 are samples , 1, 3, 5 are induced sample strains, 2, 4, 6 are uninduced sample strains. The size of the target fragment under the activation of Lac-UV5 is about 20kDa, and the red arrow in Figure 8 indicates the target fragment.

Preparation of electroporated cells: Add 1% of fresh E.coli DH5a bacteria to SOB liquid culture medium, culture at 37°C for 8-10 hours until the OD is between 0.8-1.0, then add 1ml of the bacteria solution to 100ml of sterilized SOC culture medium Cultivate at 25°C-37°C, try the best culture temperature, when the OD is 0.6, collect 100ml of bacteria, centrifuge at 3000g for 5min at 4°C. Remove the supernatant, wash the cells with 10% glycerol, centrifuge at 5000g for 5min at 4°C, and repeat twice. Use 400ul10% glycerol to suspend the bacteria, and the above steps are all performed on ice.

Electroporation of random zinc finger expression vector pBD-P-Gal11p-ZFS:

Add pBD-P-Gal11p-ZFS to the prepared competent state, mix gently, and operate in ice. Electric shock 1800V, 5ms. Place on ice for 6 minutes after electric shock, then add 800uL SOC liquid medium, incubate at 37°C, 120rpm for 1 hour, spread evenly on a plate containing chloramphenicol (34mg/ul), and electroporate 20 tubes in total, each tube electrotransformed into bacteria Spread on two 150mm Petri dishes. Inverted culture at 37°C, and finally 30 plates of transformed bacteria were obtained.

Collect all the single clones in the culture dish, extract the plasmid, and use the plasmid extraction kit from Omega Company, and refer to the instruction manual for specific steps. All the obtained plasmids were mixed to obtain a random zinc finger protein library. The obtained random zinc finger protein library is a mixture of zinc finger expression vector plasmids containing random tripartite zinc finger protein sequences.

The zinc finger protein sequence of the random zinc finger protein library is as follows:

ggggagcgcc ccttccagtg tcgcatttgc atgcggaact tttcgvnsvn mvnmvnwctt

vnwvnmcata cccgtactca taccggtgaa aaaccgtttc agtgtcggat ctgtatgcga

aatttctccv nkvnmvnsvn mttgvnwvnk catctacgta cgcacaccgg cgagaagcca

ttccaatgcc gaatatgcat gcgcaacttc agtvnkvnmv nkvnsctgvn kvnmcaccta

aaaacccacc tgaggggatc ctctgcac

Among them, N stands for A, T, C, G; V stands for G, A, C; S stands for G, C; M stands for A, C;

W stands for A, T; K stands for G, T;

Since the zinc finger protein has a conserved sequence, its nucleic acid-binding sites are -1, 1, 2, 3, 5, and 6, but these amino acids usually do not contain aromatic amino acids. By designing random primers and using overlapping PCR method to obtain three segments of zinc finger protein in its -1,1,2,3,5,6 position contains 15 or 16 kinds of amino acids (except four kinds of Phe, Tyr, Trp and Cys amino acids), encompassing all possible protein sequences of a single zinc finger protein. Then, the random combination of three zinc finger protein sequences was realized by overlapping PCR, that is, the triplet zinc finger protein sequence library was completed, and then amplified by PCR, enzyme digestion, ligation, electrotransformation and extraction of plasmids to complete the random triplet zinc finger protein library . The random triplet zinc finger protein library refers to the final extracted plasmid mixture, and the library capacity of the random triplet zinc finger protein library is greater than 1.48×10 ²¹ (15 ¹⁸ ).

Claims (2)

1. a zinc finger protein insertion vector, is characterized in that, comprises promotor and antibiotic-screening gene, in the downstream of promotor, contains Gal11p gene order, contains zinc finger protein sequence insertion point in the downstream of Gal11p gene order;

Described zinc finger protein insertion vector is pBD-P-Gal11p-ZFR, by restriction enzyme site, lac-UV5 promotor is cloned into carrier pBD, then Gal11P gene order is cloned into the downstream of lac-UV5 promotor, then the insertion point ZFR of zinc finger protein sequence is cloned into the downstream of Gal11p gene order;

The nucleotide sequence of lac-UV5 promotor is as shown in SEQ.ID.NO.1; The nucleotide sequence of Gal11P gene order is as shown in SEQ.ID.NO.2; The nucleotide sequence of zinc finger protein sequence insertion point is as shown in SEQ.ID.NO.3.

2. a random zinc finger protein expression vector, it is characterized in that, be by the random zinc finger protein sequence as shown in SEQ.ID.NO.4 with sticky end, by BbsI restriction enzyme site, be inserted in the insertion point ZFR of zinc finger protein sequence of pBD-P-Gal11p-ZFR carrier;

Described pBD-P-Gal11p-ZFR carrier,, by restriction enzyme site, lac-UV5 promotor is cloned into carrier pBD, then Gal11p gene order is cloned into the downstream of Lac-UV5 promotor, then the insertion point ZFR of zinc finger protein sequence is cloned into the downstream of Gal11p gene order and forms;

The nucleotide sequence of lac-UV5 promotor is as shown in SEQ.ID.NO.1; The nucleotide sequence of Gal11P gene order is as shown in SEQ.ID.NO.2; The nucleotide sequence of zinc finger protein sequence insertion point is as shown in SEQ.ID.NO.3.