CN102660642A - Screening system for screening zinc finger protein - Google Patents

Abstract

The invention discloses a screening system for screening zinc finger protein. The screening system comprises two reporting carriers, namely pReport-N1-LacZ and pReport-N2-EGFP and one activate carrier, namely pAD-RNAP-alpha-GAL4. By constructing matching of three elements, constructing two sets of screening systems and by using the matching of the three elements, the relevance of zinc finger protein and zinc finger nuclease recognition sequence specificity recognition and report gene expression indication is achieved, and the activity of zinc finger protein screening is improved through interactive screening of the two sets of systems. Interactive verification of the two sets of systems improves specificity in zinc finger protein screening.

Description

A screening system for screening zinc finger proteins

technical field

The invention belongs to the technical field of screening zinc finger proteins, and relates to a screening system for screening zinc finger proteins.

Background technique

Transgenic animals have broad application prospects. Gene targeting technology can achieve site-specific integration of exogenous genes, and its expression can be precisely regulated without affecting the regulation of host bacteria's own gene expression. Gene targeting mainly relies on homologous recombination. The probability of homologous recombination under natural conditions is very low, about one in a million, which greatly limits the wide application of this technology. How to improve the efficiency of gene targeting has become the focus and focus of current research.

Zinc finger proteins are a class of transcription factors with finger-like domains, which play an important role in the regulation of gene expression, cell differentiation, embryonic development, and enhancement of plant stress resistance. In 1996, Kim et al. found that the known zinc finger structure for recognizing specific DNA sequences and the non-specific recognition cleavage domain of the restriction endonuclease FokI can be combined into a new restriction enzyme through a "linker" fusion expression. Sexual endonuclease-zinc finger nuclease (zinc finger nuclease, ZFN), the recognition and cutting site is determined by the zinc finger structure.

ZFNs are composed of two parts, including a DNA-binding domain and a nonspecific endonuclease. Its working principle is that the DNA-binding domain binds to a specific DNA structure, and the non-specific endonuclease connected to it then performs a cleavage effect, causing a break at the binding site, promoting homologous recombination, and increasing the frequency of site-directed mutation and replacement. Studies have shown that gene targeting efficiency can be improved by specific zinc finger nucleases. The key to zinc finger nucleases is to screen zinc finger proteins with high specificity and high affinity.

The methods for obtaining zinc finger proteins mainly include screening method and module assembly method. 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 has higher reliability, but it is more time-consuming than the module assembly method.

Contents of the invention

The problem to be solved by the present invention is to provide a screening system for screening zinc finger proteins, which uses a reporter carrier to replace the existing bacterial screening system that must integrate the sequence into the bacterial genome for screening, and the screening process is efficient and simple.

The present invention is achieved through the following technical solutions:

A screening system for screening zinc finger proteins, comprising two reporter vectors pReport-N1-LacZ, pReport-N2-EGFP and one activation vector pAD-RNAP-alpha-GAL4;

The reporter vector pReport-N1-LacZ includes a zinc finger nuclease recognition sequence insertion site, a ribosome binding site and a promoter, and a reporter gene LacZ and an antibiotic screening gene are arranged downstream of the promoter;

The reporter vector pReport-N2-EGFP includes a zinc finger nuclease recognition sequence insertion site, a ribosome binding site and a promoter, and a reporter gene EGFP and an antibiotic screening gene are arranged downstream of the promoter;

The activation vector pAD-RNAP-alpha-GAL4 includes RNA polymerase alpha and activation element GAL4.

The nucleotide sequence of the zinc finger nuclease recognition sequence insertion site, ribosome binding site and promoter of the reporter vector pReport-N1-LacZ is shown in SEQ.ID.NO.1;

The nucleotide sequence of the zinc finger nuclease recognition sequence insertion site, ribosome binding site and promoter of the reporter vector pReport-N2-EGFP is shown in SEQ.ID.NO.2.

Both the reporting vectors pReport-N1-LacZ and pReport-N2-EGFP are provided with single-copy control elements.

The antibiotic screening gene in the reporter vector pReport-N1-LacZ is the aada gene, and the reporter gene LacZ and the aada gene are connected through a tandem expression sequence;

The antibiotic screening gene in the reporter vector pReport-N2-EGFP is the aada gene, and the reporter gene EGFP and the aada gene are connected through a tandem expression sequence;

The nucleotide sequence of the tandem expression sequence is shown in SEQ.ID.NO.3.

The RNA polymerase α in the activation vector pAD-RNAP-alpha-GAL4 is connected with the activation element GAL4 by expressing a linker sequence of five amino acid residues.

The five amino acid residues are Gly-Ser-Ala-Ala-Ala.

The reporting vector pReport-N1-LacZ and the reporting vector pReport-N2-EGFP also insert the zinc finger nuclease recognition sequence insertion site through the zinc finger nuclease recognition sequence insertion site.

The zinc finger protein recognition sequence is one shown in SEQ.ID.NO.4 to SEQ.ID.NO.9.

The reporter vector pReport-N1-LacZ and the activation vector pAD-RNAP-alpha-GAL4 are co-transfected into host bacteria to form a LacZ reporter system;

The reporter vector pReport-N2-EGFP and the activation vector pAD-RNAP-alpha-GAL4 are co-transfected into host bacteria to form an EGFP reporter system.

The random zinc finger library plasmids to be screened with random zinc finger proteins and GAL11P elements were transfected in the LacZ reporter system and the EGFP reporter system respectively; then antibiotics were added to screen positive clones, and the zinc finger proteins were screened out according to the expression of the reporter gene Then, the zinc finger protein particles screened by the LacZ reporter system were screened by the EGFP reporter system, and the zinc finger protein particles screened by the EGFP reporter system were screened by the LacZ reporter system.

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

The screening system for screening zinc finger proteins provided by the present invention is a zinc finger protein suitable for matching and identifying zinc finger nuclease recognition sequences from a random zinc finger plasmid library. By constructing the cooperation of three elements, two A set of screening systems, and then through the interactive screening of these two systems to improve the activity of zinc finger protein screening.

The coordination of the three elements is cleverly used to realize the specific recognition of zinc finger protein and zinc finger nuclease recognition sequence and the correlation of reporter gene expression indication:

The promoter in the reporter system includes the insertion site and the promoter that starts the follow-up reporter gene. When activated, the follow-up Laz reporter gene and EGFP reporter gene are started to realize different reports;

RNAP-alpha GAL4 in the activation system, wherein GAL4 is the activation element, and RNAP is the promoter in the start-up reporter system;

In the random zinc finger library plasmids to be screened, the zinc finger protein mainly binds to the zinc finger nuclease recognition sequence, and Gal11p is also an activation element;

When the set random zinc finger library plasmid is transfected into the host bacteria containing both the reporter vector and the activation vector, if the binding site of the zinc finger protein to be screened in the reporter vector can be combined with one of the random zinc finger plasmids Zinc finger protein interaction activates GAL4 on the carrier to approach and bind to the GAL11P element that binds to the zinc finger protein sequence on the random zinc finger library plasmid, and the combination of GAL4 and GAL11P prompts RNA polymerase α to bind to the promoter of the reporter vector region to activate the expression of the reporter gene LacZ or EGFP; if the recognition site to be screened has no interaction with the zinc finger protein, the expression of the reporter gene cannot be activated.

The mutual verification of the two systems is the single clone obtained through the screening of the LacZ reporter system, the extracted plasmid is transferred into the EGFP system to identify EGFP activity; refers to the single clone obtained through the screening of the EGFP reporter system, the extracted plasmid is transferred into the LacZ system to identify the LacZ activity ; Improved specificity for screening zinc finger proteins by reporting from both systems.

The screening system for screening zinc finger proteins provided by the present invention has the characteristics of large screening capacity, high efficiency, and low toxicity: first, the screening system uses the single-copy nature of the reporter carrier, replacing the previous bacterial screening system that must integrate the sequence into the bacterial genome for screening The method, the screening process is efficient and concise, the bacteria grow quickly, and the transformation efficiency is high, so a large number of interactions can be detected simply and quickly; secondly, the use of bacteria as hosts avoids yeast as hosts, and its host proteins affect the interaction between target proteins Effect; at the same time, the transfer of eukaryotic proteins into yeast will cause toxicity to the host, and the transfer of eukaryotic proteins into bacteria will reduce the generation of cytotoxicity.

The screening system for screening zinc finger proteins provided by the present invention takes the zinc finger nuclease recognition site on the bovine β casein gene as the target, designs the zinc finger nuclease recognition half site on the casein gene, and selects from a random zinc finger plasmid Zinc finger proteins were screened in the library, and highly specific zinc finger proteins were obtained through streptomycin screening and two reporter systems of LacZ and streptomycin and EGFP, and the zinc finger proteins obtained by screening have been tested on bovine fibroblasts Verified, it works fine.

Description of drawings

Fig. 1 is pRport-LacZ plasmid map;

Figure 2 is the electrophoresis pattern of pMD19T-T-A and pRport-N1 double digested with HindIII and AvrII;

Fig. 3 is pRport-EGFP plasmid map;

Fig. 4 is HindIII and AvrII double digestion pReport-N2 and PMD19T-T-E-A electrophoresis pattern;

Fig. 5 is pAD-R-G plasmid map;

Fig. 6 is pAD-R-G digestion and identification electrophoresis pattern;

Figure 7 is the electrophoresis pattern of the NheI and ASCI double-digested reporter vector pReport-N1-T-L-A;

Figure 8 is a PCR identification map of the reporter vector pReport-N1-T-L-A-CN inserted into the half-binding site of bovine casein;

Fig. 9 is the electrophoresis pattern of the reporter vector pReport-N2-T-E-A digested with Bsu36I;

Figure 10 is the PCR identification map of the reporter vector pReport-N2-T-E-A-CN inserted into the half-binding site of bovine casein;

Figures 11-1 to 11-2 are schematic diagrams of the combination screening system of the reporter carrier, the activation carrier and the random zinc finger plasmid;

Figure 12 shows the zinc finger proteins identified and screened by PCR.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

1. Construction of the reporter vector pReport-N1-LacZ

1.1 Design overlapping primers LWP1, LWP2, LWP3, LWP4, and obtain two restriction sites including Asc I and Nhe I through overlapping PCR (the zinc finger nuclease recognition sequence is inserted through these two restriction sites) and -35, -10 (weak promoter), lac operator (lactose promoter operon) and the sequence of RBS (ribosome binding site) basic element; Described primer is specifically:

LWP-1: gtacatgcat gctgtggaag ggcgcgcctg gctcgtaggc cgctagc;

LWP-2: cggaagcata aagtgtaaag cccggggtgc ctaatgctag cggcctacg;

LWP-3: ctttatgctt ccggctcgta tgttgtgtcg aattgtgagc ggataac;

LWP-4: cggcctaggc ataagctttt cctgtgtgaa attgttatcc gctcac.

The first round amplifies the LWP12 and LWP34 fragments respectively; the amplification conditions are respectively:

LWP14ul; LWP24ul; pfu Mix (2x) 25ul; ddH ₂ O 17ul;

LWP34ul; LWP2/LWP44ul; pfu Mix (2x) 25ul; ddH ₂ O 17ul;

The amplification programs are: 95°C for 3min; 95°C for 30s, 50°C for 30s, 72°C for 30s, 10cycle; 72°C for 10min;

The second round of amplification of the LWP1234 fragment; its amplification conditions are:

LWP1212.5ul; LWP3412.5ul; pfu Mix (2x) 12.5ul; ddH ₂ O 12.5ul;

The amplification programs are: 95°C for 3min; 95°C for 30s, 55°C for 30s, 72°C for 30s, 10cycle; 72°C for 10min;

The third round of amplification of the LWP fragment; its amplification conditions are:

LWP12341ul; LWP15ul; LWP45ul; pfu Mix 12.5ul; ddH ₂ O 12.5ul;

The amplification programs are: 95°C for 3min; 95°C for 30s, 60°C for 30s, 72°C for 30s, 30cycle; 72°C for 10min;

After the LWP fragment PCR product was electrophoresed with 2.5% agarose, the 149 bp DNA fragment was recovered with a gel recovery kit.

The vector pReport and LWP fragments were double-digested with AvrII and SphI, respectively, and ligated with T4 ligase (ligated overnight in a water bath at 16°C) to obtain the vector pReport-N1. Wherein N1 represents the promoter constructed, and its nucleotide sequence is as shown in SEQ.ID.NO.1, through which the insertion of the zinc finger nuclease recognition sequence (two restriction sites of Asc I and Nhe I) and Initiation of the lacz reporter gene.

The pReport-N1 vector was transformed into DH5α competent cells. Spread the transformation product evenly on a plate containing LB solid medium containing 100ug/ml amp, culture it upside down at 37°C for 12 hours, pick out a single colony, and select the correct single colony for sequencing after identification by colony PCR. Select a single colony with correct sequencing, culture 100ml of bacterial liquid, and extract the plasmid with the OMGA medium extraction kit.

1.2 Construct the pMD19T-T-L-A vector in series with the aada gene and lacz gene using the vector pMD19T as the basic backbone

1.2.1 Design primers TES-1 and TES-2 to amplify the TES sequence by overlapping PCR; TES-1 and TES-2 are specifically:

TES-15′GCCGTCGACTAACCGGGCAGGCCATGTCTGCCCGTATTTCG 3′

TES-25′CGCGGATCCTTTCCTTACGCGAAATACGGGCAGACATGG 3′

The amplification system of TES sequence is: pfu Mix 25ul; TES-15ul; TES-25ul; ddH ₂ O15ul;

The amplification program is: 3min at 95°C; 30s at 95°C, 30s at 57°C, 15s at 72°C, 10cycle; 4min at 72°C; the final TES sequence is shown in SEQ.ID.NO.3;

After the amplified TES sequence was recovered, the carrier PMD-19T and TES sequence were double-digested with BamH I and Sal I respectively, and connected with Solution I (Takara code D6022A) (connected overnight in a water bath at 16°C) to obtain the carrier PMD19T-T;

The PMD19T-T vector was transformed into DH5α competent cells, and the transformation product was evenly spread on a plate containing 100ug/ml amp LB solid medium, and after being inverted at 37°C for 12 hours, single colonies were picked and identified by colony PCR. Select the correct single colony for sequencing. Select a single colony with correct sequencing, culture 100ml of bacterial liquid, and extract the plasmid with the OMGA medium extraction kit.

1.2.2 Design primers Sm-F and Sm-R to amplify the aada fragment using the vector pCDFDuet-1 as a template. Sm-F and Sm-R are specifically:

Sm-F: ggggatccat gagggaagcg gtgat;

Sm-R: gctgaattcc taggtcaggc atttgagaag c;

The amplification program is: 95°C for 3min; 95°C for 45s, 53°C for 30s, 72°C for 90s, 30cycle; 72°C for 4min;

After recovering the amplified aada fragment, EcoR I and BamH I were used to double-cut the vector pMD19T-T and aada fragment respectively to insert the streptomycin expression gene (aada) downstream of the TES fragment, and connect with Solution I (Takara code D6022A) ( 16°C water bath overnight connection) to obtain the carrier PMD19T-T-A;

The PMD19T-T-A vector was transformed into DH5α competent cells, and the transformation product was uniformly spread on a plate containing LB solid medium containing 100ug/ml amp, and cultured upside down at 37°C for 12 hours, and a single colony was picked, and after identification by colony PCR, Select the correct single colony for sequencing. Select a single colony with correct sequencing, shake the bacteria, and extract the plasmid with the extraction kit.

1.2.3 Design the primer pair LacZ-F and LacZ-R, and use the vector pLenti6/V5-GW/lacZ as a template to amplify the LacZ gene. LacZ-F and LacZ-R are specifically:

LacZ-F: gggcagcga agcttatgat agatcccgtc g;

LacZ-R: cgcggtcgac ttaattatttt tgacaccaga cca;

The amplification program is: 95°C for 3min; 95°C for 45s, 60°C for 30s, 72°C for 5min, 30cycle; 72°C for 10min;

Recover the amplified LacZ, use Sal I and Sph I double-cutting vectors pMD19T-T-A and LacZ, respectively, to insert the reading frame of the LacZ gene upstream of the TES sequence, and connect with Solution I (Takara codeD6022A) (16°C water bath for overnight connection) Finally, the vector PMD19T-T-L-A was obtained;

The PMD19T-T-L-A vector was transformed into DH5α competent cells, and the transformation product was uniformly spread on a plate containing LB solid medium containing 100ug/ml amp, and cultured upside down at 37°C for 12 hours, and single colonies were picked out, and after identification by colony PCR, Select the correct single colony for sequencing. Select a single colony with correct sequencing, shake the bacteria, and extract the plasmid with the extraction kit.

1.2.3 Digest the pReport-N1 and pMD19T-T-L-A vectors with HindIII and AvrII, recover the corresponding fragments, connect the two fragments with Solution I (Takara code D6022A) (16°C water bath overnight connection) to obtain the basic reporter vector pReport-N1- LacZ (pReport-N1-T-L-A). Its plasmid map is shown in Figure 1. It can be seen that the elements constructed above are arranged as follows: N1 promoter (RBS, Asc I/Nhe I restriction site and -35, -10, lac operator,) lacZ, TES, Aada.

The constructed double restriction map of pReport-N1 and pMD19T-T-L-A is shown in Figure 2, where M is Trans15K. Lane 1 is the pMD19T-T-L-A vector plasmid; lanes 2 and 3 are the results of HindIII and AvrII double digestion of pMD19T-L-A vector, the size is 4071bp; lanes 4 and 5 are the results of HindIII and AvrII double digestion of pReport-N1, the size is 12309bp; Lane 6 is the pReport-N1 plasmid band.

The pReport-LacZ vector was transformed into DH5α competent cells, and the transformation product was uniformly spread on a plate containing LB solid medium containing 100ug/ml amp, and cultured upside down at 37°C for 12 hours, and single colonies were picked, and identified by colony PCR, Select the correct single colony for sequencing. Select a single colony with correct sequencing, shake the bacteria, and extract the plasmid with the extraction kit.

2. Construction of vector pReport-N2-EGFP

2.1 Design overlapping primers EWP-1, EWP-2, EWP-3, EWP-4, obtain the sequence of two consecutive Bsu36I restriction sites and -35, -10, lac operator and RBS basic elements by overlapping PCR, described The primers are specifically:

EWP-1: gccatcgatt gtggaagggc gcgcctggct cgtaggccgc atgc;

EWP-2: cggaagcata aagtgtaaag cccggggtgc ctaatcctaa ggggcctac;

EWP-3: ctttatgctt ccggctcgta tgttgtgtcg aattgtgagc ggataac;

EWP-4: cggcctaggc ataagctttt cctgtgtgaa attgttatcc gctcac;

In the first round, EWP12 and EWP34 fragments were amplified respectively; the amplification conditions were as follows:

EWP14ul; EWP24ul; pfu Mix (2x) 25ul; ddH ₂ O 17ul;

EWP34ul; EWP2/LWP44ul; pfu Mix (2x) 25ul; ddH ₂ O 17ul;

The amplification programs are: 95°C for 3min; 95°C for 30s, 50°C for 30s, 72°C for 30s, 10cycle; 72°C for 10min;

The second round of amplification of the EWP1234 fragment; its amplification conditions are:

EWP1212.5ul; EWP3412.5ul; pfu Mix (2x) 12.5ul; ddH ₂ O 12.5ul;

The amplification programs are: 95°C for 3min; 95°C for 30s, 55°C for 30s, 72°C for 30s, 10cycle; 72°C for 10min;

The third round of amplification of the EWP fragment; its amplification conditions are:

EWP12341ul; LWP15ul; LWP45ul; pfu Mix 12.5ul; ddH ₂ O 12.5ul;

The amplification programs are: 95°C for 3min; 95°C for 30s, 60°C for 30s, 72°C for 30s, 30cycle; 72°C for 10min;

After the EWP fragment PCR product was electrophoresed with 2.5% agarose, the 146bp DNA fragment was recovered with a gel recovery kit.

The vector pReport and EWP fragments were double-digested with AvrII and Sph I, respectively, and ligated with T4 ligase (overnight ligation in a water bath at 16°C) to obtain the vector pReport-N2. Wherein N2 represents the promoter constructed, and its nucleotide sequence is shown in SEQ.ID.NO.2, through which the insertion of the zinc finger nuclease recognition sequence (two Bsu36I restriction sites) and the EGFP reporter gene are realized start.

The pReport-N2 vector was transformed into DH5α competent cells. Spread the transformation product evenly on a plate containing LB solid medium containing 100ug/ml amp, culture it upside down at 37°C for 12 hours, pick out a single colony, and select the correct single colony for sequencing after identification by colony PCR. Select a single colony with correct sequencing, culture 100ml of bacterial liquid, and extract the plasmid with the OMGA medium extraction kit.

2.2 Construct the pMD 19T-T-E-A vector in series with the aada gene and EGFP gene using the vector pMD19T as the basic backbone

2.2.1 Design the primer pair EGFP-F and EGFP-R, and use the carrier pEGFP-C1 as a template to amplify the EGFP gene. EGFP-F and EGFP-R are specifically:

EGFP-F: gctgcatgcg aagcttagg tgagcaaggg;

EGFP-R: ggagtcgact tattacttgt acagctcgtc catgcc;

The amplification program is: 95°C for 3min; 95°C for 45s, 60°C for 30s, 72°C for 90s, 30cycle; 72°C for 10min;

Recover the amplified EGFP, use Sal I and Sph I double-cut vectors pMD19T-T-A and EGFP respectively, and connect with Solution I (16°C water bath for overnight connection), so that the upstream of the TES sequence is inserted into EGFP to obtain the vector PMD19T-T-E-A;

The PMD19T-T-E-A vector was transformed into DH5α competent cells, and the transformation product was uniformly spread on a plate containing LB solid medium containing 100ug/ml amp, and cultured upside down at 37°C for 12 hours, and a single colony was picked, and after identification by colony PCR, Select the correct single colony for sequencing. Select a single colony with correct sequencing, shake the bacteria, and extract the plasmid with the extraction kit.

2.2.2 Digest the pReport-N2 and PMD19T-T-E-A vectors with HindIII and AvrII, recover the corresponding fragments, and connect the two fragments with Solution I (16°C water bath overnight connection) to obtain the basic reporter vector pReport-N2-EGFP (pReport- N2-T-E-A). Its plasmid map is shown in Figure 2, and it can be seen that the elements constructed above are arranged as follows: N2 promoter (RBS, two Bsu36I restriction sites and -35, -10, lac operator,) EGFP, TES (tandem expression sequence), Aada.

The double-digested pReport-N2 and PMD19T-T-E-A vector maps are shown in Figure 4: where M is frans15k; lane 1 is pReport-N2 double cut by HindIII and AvrII, and its size after double cutting is 12461bp; lane 2 is PMD19T- T-E-A was double-cut by HindIII and AvrII, and its double-cut size was 1731bp.

The pReport-EGFP vector was transformed into DH5α competent cells, and the transformation product was uniformly spread on the LB solid medium plate containing 100ug/ml amp, and cultured upside down at 37°C for 12 hours, and a single colony was picked, and after identification by colony PCR, Select the correct single colony for sequencing. Select a single colony with correct sequencing, shake the bacteria, and extract the plasmid with the extraction kit.

3. Construction of activation vector pAD-RNAP-alpha GAL4 (pAD-R-G)

3.1 Use the carrier pBD-LGF2 as a template to amplify the Gal4 fragment, and the amplification primers are:

Gal4-F: ggcgcggccg cagaatcaa;

Gal4-R: ctcgaactag ttcaaaataa tcctgttaac aat;

The amplification program is: 95°C for 3min; 95°C for 30s, 57°C for 45s, 72°C for 45s, 35cycle; 72°C for 10min;

The amplified Gal4 was recovered, and Not I and Spe I were used to double-cut the vectors pTRG and Gal4, respectively, and T4 DNA ligase was ligated overnight at 16°C to obtain the vector pTRG-R-G;

The pTRG-R-G vector was transformed into DH5α competent cells, and the transformation product was evenly spread on the plate containing LB solid medium containing 100ug/ml amp, and cultured upside down at 37°C for 12 hours, and single colonies were picked, identified by colony PCR, and selected Correct single colony sequencing. Select a single colony with correct sequencing, shake the bacteria, and extract the plasmid with the extraction kit.

3.2 Use the pTRG-R-G vector as a template to amplify RNAP-alpha GAL4, and the amplification primers are:

28R-F: ctcgaattca aataagtgcc ttcccatca;

28R-R: gcgagatctc gctcagtgga acgaaaa;

The amplification program is: 95°C for 3min; 95°C for 45s, 60°C for 45s, 72°C for 90s, 30cycle; 72°C for 10min;

Recover the amplified RNAP-alpha GAL4, use BglII and NcoI to double-cut the vector pAD and RNAP-alpha GAL4, and T4 DNA ligase to ligate overnight at 16°C to obtain the vector pAD-R-G; its plasmid map is shown in Figure 5, you can see To the target element RNAP-alpha GAL4 cloned into the corresponding position. The results of enzyme digestion identification are shown in Figure 6: M1 is Trans 2k plus DNA Marker; M2Trans 15k DNA Marker; 1 and 2 are the results of two different monoclonal digestions of the pAD-R-G activation vector, and the size of the two fragments is 5582bp and 885bp.

4. In order to verify the above two reporter vectors, specifically targeting the zinc finger nuclease recognition site on the bovine β casein gene, design the following zinc finger nuclease recognition half-sites on the casein gene:

Specifically design the following three zinc finger nuclease recognition sites CNS1, CNS2, and CNS3 (respectively shown in SEQ.ID.NO.4-9). Each recognition site includes left and right half-recognition sites, and the underlined part is the sequence of the left and right two half-binding sites.

CNS1: 1882TAGAAGCAACTATAAA GATTTGTAA C 1907

1882A TCTTCGTTG ATATTTTCTAAACATTG 1907

CNS2 : 3706CATCCTTGCCTGCCTGGTGGCTCTG 3730

3706G TAGGAACGG ACGGACCACCGAGAC 3730

CNS3: 3723TGGCTCTGGCCCTTGC AAGAGAGGT A3748

3723A CCGAGACCG GGAACGTTCTTCTCAT3748

Six half-site primers on the three sites of CNS1, CNS2, and CNS3 were synthesized, and six half-site insertion sequences were obtained by annealing: CNS1Y, CNS1Z, CNS2Y, CNS2Z, CNS3Y, and CNS3Z.

Wherein, Y represents the half-recognition site on the right side of the zinc finger nuclease, and Z represents the half-recognition site on the left side of the zinc finger nuclease.

Insert the zinc finger nuclease recognition half-site on the casein gene through Asc I and Nhe I into the pReport-N1-T-L-A reporter vector to obtain the reporter vector pReport-N1-T-L-A-CN, where CN represents CNS1Y, CNS1CNS2Y , CNS2Z, CNS3Y or CNS3Z;

Asc I and Nhe I double-cut pReport-N1-T-L-A reporter vector is shown in Figure 7: where M is trans15k, 1 is the plasmid, and 2 is the double restriction band with a size of 16360bp.

The PCR identification report vector pReport-N1-T-L-A-CN is shown in Figure 8: where M is trans5k, 1-22 is the picked monoclonal PCR identification band, the band size is 750bp, its 1, 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 19, 20 are positive clones.

At the same time, insert the zinc finger nuclease recognition half site on the casein gene through two Bsu36I restriction sites into the pReport-N2-T-E-A reporter vector to obtain pReport-N2-T-E-A-CN, where CN represents CNS1Y, CNS1CNS2Y , CNS2Z, CNS3Y or CNS3Z;

The pReport-N2-T-E-A reporter vector digested with Bsu36I is shown in Figure 9: M is trans15k; 1 is the pReport-N2-EGFP Bsu361 restriction map, and its size is 14142bp; 2 is the plasmid pReport-N2-EGFP.

The PCR identification of pReport-N2-T-E-A-CN is shown in Figure 10: where M is trans2Kplus; 1, 2 are PCR identification bands, and the size of the target band is 750bp. The center of the half-site is located at the transcription start-62, which is the best binding site.

5. In order to better realize the screening of zinc finger proteins, the interacting Gal11p and Gal4 were respectively set in the random zinc finger library plasmid and the activation vector. 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.

As shown in Figures 11-1 and 11-2, when the set random zinc finger library plasmid is transfected into the host bacteria containing the reporter vector and the activation vector, if the zinc finger protein to be screened located in the reporter vector binds The site can interact with a certain zinc finger protein in the random zinc finger plasmid, then activate GAL4 on the vector and the GAL11P element that binds to the zinc finger protein sequence on the random zinc finger library plasmid to approach and combine, after GAL4 binds to GAL11P Promote the binding of RNA polymerase α to the promoter region of the reporter vector to activate the expression of the reporter gene LacZ or EGFP (as shown in Figure 11-1); if the recognition site to be screened has no interaction with the zinc finger protein, the reporter gene cannot be activated Expression (shown in Figure 11-2).

6. Screening and condition optimization of zinc finger proteins

1) Transfer the pAD-R-G vector into DH5a to prepare competent cells (refer to the method for preparing supercompetent cells in the second edition of molecular cloning), and then transfer a part of them into the pReport-N1-T-L-A-CN reporter vector to obtain cells containing two plasmids The other part of strain DH5a-L was transformed into pReport-N2-T-E-A-CN reporter vector to obtain strain DH5a-E containing two plasmids.

2) Use the strain DH5a-L and the strain DH5a-E containing the two plasmids to prepare electrotransformed cells: inoculate 1% of fresh strains in SOB liquid culture medium, and culture at 37°C for 8-10 hours until the OD is between 0.8-1.0 Occasionally, inoculate 1ml of bacterial liquid in 100ml of sterilized SOC culture medium and cultivate at 25°C-37°C. When the OD is 0.6, collect 100ml of bacterial cells, centrifuge at 3000g for 5min at 4°C, remove the supernatant, and wash the bacterial cells with 10% glycerol , 4°C, centrifuge at 5000g for 5min, repeat twice, suspend the bacteria with 400ul10% glycerol, and all the above steps are performed on ice.

3) Electroporation conditions: Add the zinc finger protein library plasmid concentration of 10ug to 20ug per 1ul as a gradient into the prepared electroporation competent cells, 1500-2500v electric shock for 5ms, place on ice for 6min-8min, add SOC culture medium, Shake the bacteria at 37°C and 120rpm for 1 hour, and apply the bacteria obtained from the shaking to the solid medium of various antibiotics for screening. Adjust the concentration of various antibiotics, adjust the amount of glucose in the culture medium and culture medium. Electric shock cup specifications: Gap (mm) 2.0, Minimum Volume 40μl, Maximum Volume 400μl. Electrotransform the random zinc finger library, add the zinc finger library plasmid into the prepared electrotransformed cells, and operate on ice. Electric shock 1800v-2500v, 5ms.

Place on ice for 6 minutes after electric shock, then add SOC liquid culture medium, culture at 37°C, 120rpm for 1h, then coat on solid medium containing various antibiotics, and culture at 37°C upside down. Pick a single clone for identification.

And X-gal should be added to the LacZ reporter system to display the activity of LacZ.

4) Results of screening and condition optimization of zinc finger proteins:

The optimal shaking temperature for preparing electrotransformation competent cells is 26.7°C, and the optimal electric shock condition for electrotransformation is 2000v, 5ms, and the cell damage is minimal.

Determine the final antibiotic concentration kan is 30ug/m; Cm is 34ug/ml; Amp is 50ug/ml; Sm concentration<80ug/ml, 0.2%gluclose, IPTG is 50uM.

5) After reporting the expression of the reporter vector, the zinc finger protein obtained by PCR identification and screening is shown in Figure 12: wherein M is a 50bp marker (from bottom to top is 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 400bp, 500bp ) 1-7 is the PCR result of strain plasmid obtained by screening, and the fragment size is 273bp.

6) Determination of zinc finger protein EGFP/LacZ activity

The anti-streptomycin concentration of the zinc finger protein obtained by screening was as high as 80ug/ml, and the EGFP/LacZ activity of the obtained zinc finger protein was determined. The resulting monoclonal plasmid was extracted, and random zinc finger protein particles with high purity were obtained through two rejoining and two retransformation, and the zinc finger protein particles screened by the LacZ system were transferred to the EGFP system, and the EGFP intensity was determined; the EGFP system The screened zinc finger proteins were transfected into LacZ system to measure their LacZ activity. The interactive use of the two systems strengthens the reliability of the zinc finger proteins screened. The zinc finger proteins screened were identified by PCR and sequencing.

Wherein the method of Lac activity assay: pick the monoclonal that screens and obtain from the glass dish, inoculate with various antibiotics (kan is 30ug/m; Cm is 34ug/ml; Amp is 50ug/ml; the concentration of Sm is 40ug/ml; ml) of soc culture medium, shake overnight at 37°C and 200 rpm. Inoculate 100 ul of the overnight shaken bacterial solution into fresh soc culture solution at 37°C and shake at 200 rpm until the OD reaches about 0.8. Collect the bacteria (centrifuge at 12000rpm for 1min), wash the bacteria twice with Z Buffer to remove the residual culture solution, dilute the bacteria so that their od600 is around 0.8, add 200ul of the obtained bacteria solution into a 96-well plate and use an od instrument Accurately measure the OD600 value of each bacterial solution, use the pReprorT-N1-LacZ plasmid strain as the control group, and use the Z Buffer solution as the error control group. Take 100 ul of bacterial liquid and add 11 ul of Lysis Solution A, and lyse for 15 min at room temperature. Mix 135ul of Z Buffer containing mercaptoethanol and 30ml of 4mg/ml ONPG into a 9-well plate, then add 15ul of the obtained lysate, and mix well. The dynamic method was used to measure the OD420 value, and the change of the OD420 value within 50 minutes was measured every 50 seconds. The Z Buffer containing mercaptoethanol and ONPG mixture was used as the control group. The obtained OD420 value was plotted on the time axis, and the slope of the obtained trend line was the cleavage rate (V) of ONPG, and the final LacZ activity was obtained by using the formula LacZ activity=V*1000/OD600. The measured lacz activity is shown in Table 1. Compared with the control group, the zinc finger protein obtained by screening in the experimental group significantly increased the activity of lacz.

Table 1 The activity of lacz of the zinc finger protein screened

control CSNY1 CSNY5 CSNY9 CNSZ3 CNSZ4 CNSZ16 OD600 0.773 0.796 0.673 0.788 0.77 0.888 0.865 velocity 0 0.0101 0.0108 0.0111 0.01 0.0114 0.0096 activity 0 12.74 16.05 14.10 13.0 12.84 11.10

And the method of EGFP intensity measurement: pick the monoclonal that screens and obtain from the glass dish, inoculate and contain various antibiotics (kan is 30ug/m; Cm is 34ug/ml; Amp is 50ug/ml; Sm concentration is 40ug/ml; ml) of soc culture medium, shake overnight at 37°C and 200 rpm. Inoculate 100 ul of the overnight shaken bacterial solution into fresh soc culture solution at 37°C and shake at 200 rpm until the OD reaches about 0.8. Collect the bacteria (centrifuge at 12000rpm for 1min), wash the bacteria twice with sodium phosphate to remove the residual culture solution, dilute the bacteria so that their od600 is about 0.5, and add 200ul of the obtained bacteria solution into a 96-well microplate plate for use The microplate reader accurately measured the OD600 value of each bacterial solution, the reporter carrier strain pReprorT-EGFP was used as the control group, and the sodium phosphate solution was used as the error control group. Then measure its fluorescence intensity, the determination condition of fluorescence intensity, adopt microplate reader to set the wavelength of fluorescence excitation light as 491nm, the emission wavelength of fluorescence as 511nm, the determination of EGFP intensity is carried out under 22 ℃. The measured fluorescence intensity/OD600 is the fluorescence intensity of the final strain. The measured EGFP activity is shown in Table 2. Compared with the control group, the zinc finger protein obtained by screening in the experimental group significantly increased the expression of EGFP.

Table 2 The activity of EGFP of the zinc finger protein screened

The screening system constructed above was used to screen the zinc finger nuclease binding site on the bovine β-casein gene, and a high-efficiency zinc finger protein was obtained. The activity of the screened zinc finger protein was verified by antibiotic screening, lacz activity measurement and high expression of EGFP, and the screened zinc finger protein has been verified on bovine fibroblasts, and the effect is good.

Claims (10)

1. A screening system for screening zinc finger proteins, characterized in that it comprises two reporter vectors pReport-N1-LacZ, pReport-N2-EGFP and an activation vector pAD-RNAP-alpha-GAL4; The reporter vector pReport-N1-LacZ includes a zinc finger nuclease recognition sequence insertion site, a ribosome binding site and a promoter, and a reporter gene LacZ and an antibiotic screening gene are arranged downstream of the promoter; The reporter vector pReport-N2-EGFP includes a zinc finger nuclease recognition sequence insertion site, a ribosome binding site and a promoter, and a reporter gene EGFP and an antibiotic screening gene are arranged downstream of the promoter; The activation vector pAD-RNAP-alpha-GAL4 includes RNA polymerase alpha and activation element GAL4. 2. The screening system for screening zinc finger proteins according to claim 1, wherein the zinc finger nuclease recognition sequence insertion site, ribosome binding site and promoter of the reporter vector pReport-N1-LacZ The nucleotide sequence is shown in SEQ.ID.NO.1; The nucleotide sequence of the zinc finger nuclease recognition sequence insertion site, ribosome binding site and promoter of the reporter vector pReport-N2-EGFP is shown in SEQ.ID.NO.2. 3 . The screening system for screening zinc finger proteins according to claim 1 , wherein the reporting vectors pReport-N1-LacZ and pReport-N2-EGFP are both provided with a single-copy control element. 4 . 4. the screening system for screening zinc finger proteins as claimed in claim 1, is characterized in that, the antibiotic selection gene in described reporter carrier pReport-N1-LacZ is aada gene, and reporter gene LacZ and aada gene pass through tandem expression sequence connected; The antibiotic screening gene in the reporter vector pReport-N2-EGFP is the aada gene, and the reporter gene EGFP and the aada gene are connected through a tandem expression sequence; The nucleotide sequence of the tandem expression sequence is shown in SEQ.ID.NO.3. 5. The screening system for screening zinc finger proteins according to claim 1, wherein the RNA polymerase α in the activation vector pAD-RNAP-alpha-GAL4 and the activation element GAL4 express five amino acid residues The linker sequence connection. 6. The screening system for screening zinc finger proteins according to claim 5, wherein the five amino acid residues are Gly-Ser-Ala-Ala-Ala. 7. The screening system for screening zinc finger proteins according to claim 1, characterized in that, in the reporter vector pReport-N1-LacZ and the reporter vector pReport-N2-EGFP, the zinc finger nuclease recognition sequence insertion position is also used Dot insertion of the zinc finger nuclease recognition sequence. 8. The screening system for screening zinc finger proteins according to claim 7, wherein the zinc finger protein recognition sequence is one shown in SEQ.ID.NO.4 to SEQ.ID.NO.9 . 9. The screening system for screening zinc finger proteins according to claim 7, characterized in that, the reporter vector pReport-N1-LacZ and the activation vector pAD-RNAP-alpha-GAL4 are co-transfected into the host bacterium to form a LacZ reporting system; The reporter vector pReport-N2-EGFP and the activation vector pAD-RNAP-alpha-GAL4 are co-transfected into host bacteria to form an EGFP reporter system. 10. The screening system for screening zinc finger proteins as claimed in claim 7, wherein the random zinc finger library that is provided with random zinc finger proteins and GAL11P elements to be screened is transfected in the LacZ reporter system and the EGFP reporter system respectively Then add antibiotics to screen positive clones, and screen the zinc finger protein plasmids according to the expression of the reporter gene; then screen the zinc finger protein plasmids screened by the LacZ reporter system through the EGFP reporter system, and screen the zinc finger protein plasmids screened by the EGFP reporter system Protein particles were screened by the LacZ reporter system.

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