CN113564141B

CN113564141B - Single-cell genome amplification enzyme mutant and application thereof

Info

Publication number: CN113564141B
Application number: CN202110840919.1A
Authority: CN
Inventors: 张佳; 苏晓璐; 朱文轩; 王晓航; 贾荟; 徐健
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2023-08-11
Anticipated expiration: 2041-07-23
Also published as: CN113564141A

Abstract

The invention discloses a mutant of single-cell genome amplification enzyme (phi 29 DNA polymerase) and application thereof. The protein is obtained by mutating phi29 DNA polymerase, the amino acid sequence of the phi29 DNA polymerase is shown as SEQ ID No.4, and the mutation is that phenylalanine at 136 th position of the phi29 DNA polymerase is mutated into cysteine and alanine at 376 th position of the phi29 DNA polymerase is mutated into cysteine. The amplification capability of phi29 DNA polymerase is improved by mutating F at 136 th position of phi29 DNA polymerase to C and mutating A at 376 th position of phi29 DNA polymerase to C to form disulfide bond.

Description

Single-cell genome amplification enzyme mutant and application thereof

Technical Field

The invention belongs to the field of biotechnology. In particular, the invention relates to mutants of single cell genomic amplification enzyme (phi 29 DNA polymerase) and uses thereof.

Background

Single-cell genome sequencing is a technology for amplifying and sequencing whole genome at a single-cell level, and along with the continuous development of a second-generation sequencing technology, the continuous reduction of sequencing cost is realized, and the rapid whole genome DNA sequence analysis and genotyping are greatly promoted by a large-scale whole genome sequencing technology. Compared with the traditional whole genome sequencing, single-cell whole genome sequencing can more accurately find SNP with low mutation frequency, has more remarkable significance for researching cell heterogeneity, and has the advantages of omnibearing and multilevel. The journal of science in 2013 lists single cell sequencing as the six major fields of prime concern in the year, and the journal of natural methodology lists single cell sequencing as the most important methodology development in 2013.

Phi29 DNA polymerase is receiving much attention because it catalyzes a key step in Whole Genome Amplification (WGA) during single cell sequencing. It mediates multiple strand displacement reaction (MDA), the most widely used WGA method at present. The Phi29 DNA polymerase is a viral DNA replicase, which is produced by a bacillus subtilis phageIs encoded by gene 2. The phi29 DNA polymerase-based MDA reaction shows significantly higher sensitivity, amplification efficiency and sequence fidelity, and less allele and site bias than PCR-based methods, and a single amplification can add at least 70,000 nucleotides (highest synthesis capacity in known DNA polymerases), and can synthesize DNA strands up to 100kb in length. In addition, the polymerase has 3'-5' exonuclease "correction" activity, reporting an error rate of 5x10 ^-6 Is 100 times lower than Taq DNA polymerase. However, the problems of high cost (160 yuan/reaction), non-specific amplification (18%), efficiency to be improved (84% coverage), uneven genome coverage, etc. remain one of the key bottlenecks of single cell sequencing technology. Thus, phi29 DNA polymerase has not only the need for upgrading modification itself, but also different biological fields require different properties thereof, and thus, there is a need to continuously improve the properties thereof.

Disclosure of Invention

It is an object of the present invention to provide a mutant of a single cell genome amplification enzyme.

The invention provides a protein which is obtained by mutating phi29 DNA polymerase, wherein the amino acid sequence of the phi29 DNA polymerase is shown as SEQ ID No.4, and the mutating comprises the following steps: phenylalanine (F) at position 136 of the phi29 DNA polymerase was mutated to cysteine (C), and alanine (a) at position 376 was mutated to cysteine (C).

Optionally, according to the protein described above, the mutation further comprises: methionine (M) at position 7 is mutated to arginine (R); valine (V) at position 50 to alanine (a); methionine (M) at position 96 is mutated to threonine (T); glycine (G) at position 196 to aspartic acid (D); glutamic acid (E) at position 220 is mutated to lysine (K); glutamine (Q) at position 496 is mutated to proline (P); lysine (K) at position 511 is mutated to glutamic acid (E); phenylalanine (F) at position 525 is mutated to leucine (L).

Alternatively, according to the above protein, the amino acid sequence of the protein is shown at positions 109-682 in SEQ ID No. 3.

The invention also provides a fusion protein, the amino acid sequence of which is the amino acid sequence of the GB1 protein connected with the N end of the amino acid sequence of the protein.

Alternatively, according to the fusion protein, the amino acid sequence of the GB1 protein is shown as 26-79 positions in SEQ ID No. 3; or the amino acid sequence of the fusion protein is shown as SEQ ID No. 3.

The present invention also provides a biomaterial, which is any one of B1) to B8):

b1 A nucleic acid molecule encoding the above protein or a nucleic acid molecule encoding the above fusion protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism containing the recombinant vector of B4).

Alternatively, according to the above biological material, the nucleic acid molecule encoding the above protein is any one of the following:

b11 Substitution of TTT at positions 406-408 and GCG at positions 1126-1128 of the optimized phi29 wild-type gene nucleotide sequence with TGC and other nucleotides with unchanged;

b12 Compared to the nucleic acid molecule according to B11), the nucleotide sequence also comprises the following substitutions:

The ATG at 19-21 bits is replaced with CGT, the GTG at 148-150 bits is replaced with GCG, the ATG at 286-288 bits is replaced with ACC, the GGT at 586-588 bits is replaced with GAC, the GAA at 658-660 bits is replaced with AAA, the CAG at 1486-1488 bits is replaced with CCG, and the AAG at 1531-1533 bits is replaced with GAA;

the nucleotide sequence of the optimized phi29 wild gene is shown in the 4 th-1725 th positions of SEQ ID No. 1.

For example, the nucleotide sequence of the nucleic acid molecule of B12) is shown at positions 109-682 of SEQ ID No. 2. The nucleotide sequence of the nucleic acid molecule for encoding the fusion protein is shown as SEQ ID No. 2.

The present invention also provides a DNA replication or amplification method comprising replicating or amplifying DNA using the above protein or the above fusion protein as a DNA polymerase.

It is a further object of the present invention to provide the use of the above proteins, the above fusion proteins and the above biological materials.

Specifically, the application of the protein and the fusion protein is any one of the following:

1) As a DNA polymerase;

2) Catalyzing DNA replication and/or catalyzing DNA amplification;

3) Catalytic rolling amplification and/or catalytic multiplex strand displacement amplification;

4) Performing DNA sequencing or RNA sequencing or whole genome sequencing;

5) RCA library establishment;

6) Genome amplification coverage detection;

7) Preparing a kit product for catalyzing DNA replication and/or catalyzing DNA amplification;

8) Preparing a product for catalytic rolling amplification and/or catalytic multiplex strand displacement amplification;

9) Preparing a product for performing DNA sequencing or RNA sequencing or whole genome sequencing;

10 Preparing a product for RCA banking;

11 Preparing a product for genome amplification coverage detection;

the application of the biological material is any one of the following:

1) Catalyzing DNA replication and/or catalyzing DNA amplification;

2) Catalytic rolling amplification and/or catalytic multiplex strand displacement amplification;

3) Performing DNA sequencing or RNA sequencing or whole genome sequencing;

4) RCA library establishment;

5) Genome amplification coverage detection;

6) Preparing a product for catalyzing DNA replication and/or catalyzing DNA amplification;

7) Preparing a product for catalytic rolling amplification and/or catalytic multiplex strand displacement amplification;

8) Preparing a product for performing DNA sequencing or RNA sequencing or whole genome sequencing;

9) Preparing a product for RCA library establishment;

10 Preparation of a product for genome amplification coverage detection.

The present invention also provides a method for producing the above protein or the above fusion protein, comprising the step of expressing a nucleic acid molecule encoding the above protein or the above fusion protein in an organism, which is a microorganism, a plant or a non-human animal, to obtain the above protein or the above fusion protein.

In the above method, expressing the nucleic acid molecule encoding the protein or the fusion protein in an organism may include introducing the nucleic acid molecule encoding the protein or the fusion protein into a recipient microorganism to obtain a recombinant microorganism expressing the protein or the fusion protein, culturing the recombinant microorganism, and expressing the protein or the fusion protein.

The nucleic acid molecule encoding the protein or the fusion protein may be introduced into a recipient microorganism, for example, a phi29005 plasmid, a phi29020 plasmid or a phi29021 plasmid prepared in the following examples.

In the above method, the recipient microorganism may be any one of C1) -C4): c1 A prokaryotic microorganism; c2 Gram-negative bacteria; c3 Bacteria of the genus escherichia; c4 Coli BL21 (DE 3).

According to the invention, the F mutation at 136 th position of phi29DNA polymerase is changed into C and the A mutation at 376 th position is changed into C to form disulfide bond, so that the amplification capability of the phi29DNA polymerase is improved. According to the embodiment of the invention, the amplification capability of the enzyme is improved through the design and construction of disulfide bonds and the addition of point mutation, the GB1 protein is recombinantly expressed to improve the soluble expression of the enzyme, and the constructed phi29DNA polymerase mutant has the advantages of high amplification efficiency, high and uniform amplification efficiency, low non-specific amplification, convenience in operation, capability of stably completing the amplification of a genome at a single cell level and the like, and the cost is reduced by hundreds of times.

Drawings

FIG. 1 is a schematic diagram of the structure of phi29021 plasmid prepared in example 1.

FIG. 2A is a graph showing the amplification results of the genome amplified by the phi29 DNA polymerase mutant of example 2 without activity.

FIG. 2B is a graph showing the amplification results of the genome amplified by the active phi29 DNA polymerase mutant of example 2;

FIG. 2C is a graph showing the results of enzyme activity measurements of the active phi29 DNA polymerase mutant of example 2.

FIG. 3 shows the results of detection of the enzyme activity and the expression level in example 3.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications.

Reagents and strains: all reagents in the present invention are commercially available reagent grade or more. Wherein, the Tryptone, yeast extract, naCl, the plasmid extraction kit, the gel recovery kit and all restriction endonucleases are all from Shanghai Bioengineering company. PrimeSTAR Max DNA polymerase, solution I ligase and pET-21c (+) vector were purchased from da Lian Bao Bio Inc. KOD Plus Neo polymerase was purchased from Shanghai Toyobo Biotechnology Co. Q5 High-Fidelity DNA polymerase was purchased from British Biotechnology Co., N.E.Beijing. As a host bacterium (Stratagene Co., calif., U.S.A.) used in DNA manipulation, E.coli BL21 (Escherichia coli BL 21) strain, luria-Bertani (LB) medium containing 100. Mu.g/ml ampicillin was used as a medium for culturing E.coli BL21.LB medium (5 g/l yeast extract, 10g/l tryptone, 5g/l NaCl) was used to induce the expression medium.

Example 1 preparation of strains:

1. construction of various plasmid vectors of the present invention:

the plasmids constructed in this example were as follows:

the pET-21c (+) -phi29 wild type plasmid is obtained by replacing a DNA fragment (the sequence is shown as SEQ ID No. 1) containing the phi29 wild type gene after codon optimization with a fragment between BamHI and Xho I recognition sites of a pET-21c (+) vector (purchased from da Lian Bao Bio-company), keeping other sequences of pET-21c (+) unchanged, and obtaining the phi29 wild type gene recombinant expression vector after codon optimization. In SEQ ID No.1, the 1 st to 3 rd positions are the start codon, the 4 th to 1725 th positions are the phi29 wild type gene after codon optimization, and the last 3 rd position is the stop codon. pET-21c (+) -phi29 wild type codon optimized vector, the expression product is phi29DNA polymerase. The amino acid sequence of phi29DNA polymerase is shown in SEQ ID No. 4.

Plasmid pZJphi29001 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by phi29-K6CA55C gene, and other nucleotides remain unchanged. The AAA (K codon) at the 16 th-18 th positions of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), the GCG (A codon) at the 163 th-165 th positions is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29001 expresses phi29-K6CA55C gene, and the expression product is phi29DNA polymerase mutant K6CA55C. The amino acid sequence of the mutant K6CA55C is that K at the 6 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, A at the 55 th position is replaced by C, and other amino acid residues are unchanged.

The plasmid pZJphi29002 differs from the pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in the pET-21C (+) -phi29 wild-type is replaced by the phi29-V23CL43C gene, and other nucleotides remain unchanged. The phi29-V23CL43C gene sequence is that the optimized phi29 wild type gene sequence is replaced by TGC (L codon) at the 67 th-69 th positions of GTT (V codon), and the CTG (L codon) at the 127 th-129 th positions is replaced by TGC (C codon) with other nucleotides unchanged. Plasmid pZJphi29002 expresses phi29-V23CL43C gene, and the expression product is phi29DNA polymerase mutant V23CL43C. The amino acid sequence of the mutant V23CL43C is that V at the 23 rd position of the amino acid sequence of phi29DNA polymerase is replaced by L, L at the 43 rd position is replaced by C, and other amino acid residues are unchanged.

The plasmid pZJphi29003 differs from the pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in the pET-21C (+) -phi29 wild-type is replaced by the phi29-Y58CS121C gene, and other nucleotides remain unchanged. The gene sequence of phi29-Y58CS121C is that the TAC (Y codon) at 172-174 th bit of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), the AGC (S codon) at 361-363 th bit is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29003 expresses phi29-Y58CS121C gene, and the expression product is phi29DNA polymerase mutant Y58CS121C. The amino acid sequence of the mutant Y58CS121C is that Y at the 58 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, S at the 121 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29004 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by phi29-E74CL405C gene, and other nucleotides remain unchanged. The GAA (E codon) at 220-222 of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), the CTG (L codon) at 1213-1215 is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29004 expresses phi29-E74CL405C gene, and the expression product is phi29DNA polymerase mutant E74CL405C. The amino acid sequence of the mutant E74CL405C is that E at the 74 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, L at the 405 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29005 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by the phi29-F136CA376C gene, and other nucleotides remain unchanged. The phi29-F136CA376C gene sequence is that TTT (F codon) at 406-418 of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), GCG (A codon) at 1126-1128 is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29005 expresses phi29-F136CA376C gene, and the expression product is phi29DNA polymerase mutant F136CA376C. The amino acid sequence of the mutant F136CA376C is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29006 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced with the phi29-S193CS387C gene, and other nucleotides remain unchanged. The gene sequence of phi29-S193CS387 is that AGC (S codon) at 577-579 of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), TCT (S codon) at 1159-1161 is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29006 expresses phi29-S193CS387C gene, and the expression product is phi29DNA polymerase mutant S193CS387C. The amino acid sequence of the mutant S193CS387C is that S at the 193 rd position is replaced by C, S at the 387 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29007 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced with the phi29-L252C D457C gene, and the other nucleotides remain unchanged. The phi29-L252CD457C gene sequence is that CTG (L codon) at 754-756 of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), GAC (D codon) at 1369-1371 is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29007 expresses phi29-L252CD457C gene, and the expression product is phi29DNA polymerase mutant L252CD457C. The amino acid sequence of the mutant L252CD457C is that L at the 252 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, D at the 457 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29008 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by the phi29-Y253CY389C gene, and other nucleotides remain unchanged. The gene sequence phi29-Y253CY389C is characterized in that the TAC (Y codon) at 757-759 positions of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), the TAC (Y codon) at 1165-1167 positions is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29008 expresses phi29-Y253CY389C gene, and the expression product is phi29DNA polymerase mutant Y253CY389C. The amino acid sequence of the mutant Y253CY389C is that Y at 253 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, Y at 389 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29009 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by the phi29-M257CF359C gene, and other nucleotides remain unchanged. The gene sequence phi29-M257C F359C is that ATG (M codon) at 769-771 of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), TTC (F codon) at 1075-1077 is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29009 expresses the phi29-M257CF359C gene, and the expression product is phi29DNA polymerase mutant M257CF359C. The amino acid sequence of the mutant M257CF359C is that M at 257 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, F at 359 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29010 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by phi29-Y280CK351C gene, and other nucleotides remain unchanged. The gene sequence of phi29-Y280CK351C is that TAC (Y codon) at 838-840 th position of the optimized phi29 wild-type gene sequence is replaced by TGC (C codon), AAA (K codon) at 1051-1053 th position is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29010 expresses phi29-Y280CK351C gene, and the expression product is phi29DNA polymerase mutant Y280CK351C. The amino acid sequence of the mutant Y280CK351C is that Y at 280 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, K at 351 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29011 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by the phi29-Y297CH338C gene, and other nucleotides remain unchanged. The gene sequence phi29-Y297CH338C is that the TAC (Y codon) at 889-891 of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), the CAT (H codon) at 1012-1014 is replaced by TGC (C codon), and other nucleotides are unchanged. Plasmid pZJphi29011 expresses phi29-Y297CH338C gene, the expression product of which is phi29DNA polymerase mutant Y297CH338C. The amino acid sequence of the mutant Y297CH338C is that Y at 297 of the amino acid sequence of phi29DNA polymerase is replaced by C, H at 338 is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29012 differs from pET-21C (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21C (+) -phi29 wild-type is replaced by phi29-V330CA434C gene, and other nucleotides remain unchanged. The gene sequence of phi29-V330CA434C is that the optimized phi29 wild type gene sequence is formed by replacing the GTG (V codon) at 988-990 th position with TGC (C codon), the GCA (A codon) at 1300-1302 th position with TGC (C codon) and other nucleotides are unchanged. Plasmid pZJphi29012 expresses phi29-V330CA434C gene, and the expression product is phi29DNA polymerase mutant V330CA434C. The amino acid sequence of the mutant V330CA434C is that V at the 330 th position of the amino acid sequence of phi29DNA polymerase is replaced by C, A at the 434 th position is replaced by C, and other amino acid residues are unchanged.

Plasmid pZJphi29013 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced with the phi29-F136CA376CM7R gene, and the other nucleotides remain unchanged. The modified phi29-F136CA376CM7R gene sequence is characterized in that the TTT (F codon) at 406 th-408 th of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), the GCG (A codon) at 1126-1128 th is replaced by TGC (C codon), the ATG (M codon) at 19-21 th is replaced by CGT (R codon), and other nucleotides are unchanged. Plasmid pZJphi29013 expresses phi29-F136CA376CM7R gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7R. The amino acid sequence of the mutant F136CA376CM7R is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, and other amino acid residues are unchanged.

Plasmid pZJphi29014 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by the phi29-F136CA376CM7RV50A gene, and other nucleotides remain unchanged. The phi29-F136CA376CM7RV50A gene sequence is characterized in that the optimized phi29 wild type gene sequence is replaced by TGC (C codon) at 406 th to 408 th positions, GCG (A codon) at 1126 th to 1128 th positions is replaced by TGC (C codon), ATG (M codon) at 19 th to 21 th positions is replaced by CGT (R codon), GTG (V codon) at 148 th to 150 th positions is replaced by GCG (A codon), and other nucleotides are unchanged. Plasmid pZJphi29014 expresses phi29-F136CA376CM7RV50A gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50A. The amino acid sequence of the mutant F136CA376CM7RV50A is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, and other amino acid residues are unchanged.

Plasmid pZJphi29015 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by phi29-F136CA376CM7RV50AM96T gene, and other nucleotides remain unchanged. The phi29-F136CA376CM7RV50AM96T gene sequence is characterized in that the optimized phi29 wild type gene sequence is replaced by TGC (C codon) at 406 th to 408 th TTT (F codon), TGC (C codon) at 1126 th to 1128 th GCG (A codon), CGT (R codon) at 19 th to 21 th ATG (M codon), GCG (A codon) at 148 th to 150 th GTG (V codon) and ACC (T codon) at 286 th to 288 th ATG (M codon), and other nucleotides are unchanged. Plasmid pZJphi29015 expresses phi29-F136CA376CM7RV50AM96T gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50AM96T. The amino acid sequence of the mutant F136CA376CM7RV50AM96T is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, M at 96 th position is replaced by T, and other amino acid residues are unchanged.

Plasmid pZJphi29016 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by phi29-F136CA376CM7RV50AM96TG196D gene, and other nucleotides remain unchanged. The phi29-F136CA376CM7RV50AM96TG196D gene sequence is characterized in that the optimized phi29 wild type gene sequence is replaced by TGC (C codon) at 406 th to 408 th TTT (F codon), TGC (C codon) at 1126 th to 1128 th GCG (A codon), CGT (R codon) at 19 th to 21 th ATG (M codon), GCG (A codon) at 148 th to 150 th GTG (V codon), ACC (T codon) at 286 th to 288 th ATG (M codon) and GAC (D codon) at 586 th to 588 th GGT (G codon) at other nucleotides are unchanged. Plasmid pZJphi29016 expresses phi29-F136CA376CM7RV50AM96TG196D gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50AM96TG196D. The amino acid sequence of the mutant F136CA376CM7RV50AM96TG196D is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, M at 96 th position is replaced by T, G at 196 th position is replaced by D, and other amino acid residues are unchanged.

Plasmid pZJphi29017 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by phi29-F136CA376CM7RV50AM96TG196DE220K gene, and other nucleotides remain unchanged. The phi29-F136CA376CM7RV50AM96TG196DE220K gene sequence is that TTT (F codon) at 406 th to 408 th of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), GCG (A codon) at 1126 th to 1128 th is replaced by TGC (C codon), ATG (M codon) at 19 th to 21 th is replaced by CGT (R codon), GTG (V codon) at 148 th to 150 th is replaced by GCG (A codon), ATG (M codon) at 286 th to 288 th is replaced by ACC (T codon), GGT (G codon) at 586 th to 588 th is replaced by GAA (E codon) at 658 th to 660 th of GAC (D codon), and other nucleotides are unchanged. Plasmid pZJphi29017 expresses phi29-F136CA376CM7RV50AM96TG196DE220K gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50AM96 TG196DE220K. The amino acid sequence of the mutant F136CA376CM7RV50AM96TG196DE220K is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, M at 96 th position is replaced by T, G at 196 th position is replaced by D, E at 220 th position is replaced by K, and other amino acid residues are unchanged.

Plasmid pZJphi29018 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by the phi29-F136CA376CM7RV50AM96TG196DE220KQ496P gene, the other nucleotides remaining unchanged. The phi29-F136CA376CM7RV50AM96TG196DE220KQ496P gene sequence is that TTT (F codon) at 406 th to 408 th of the optimized phi29 wild type gene sequence is replaced by TGC (C codon), GCG (A codon) at 1126 th to 1128 th is replaced by TGC (C codon), ATG (M codon) at 19 th to 21 th is replaced by CGT (R codon), GTG (V codon) at 148 th to 150 th is replaced by GCG (A codon), ATG (M codon) at 586 th to 288 th is replaced by ACC (T codon), GGT (G codon) at 586 th to 588 th is replaced by GAA (E codon) at 658 th to 660 th of GAC (D codon), CAG (Q codon) at 1486 th to 1488 th is replaced by CCG (P codon), and other nucleotides are unchanged. Plasmid pZJphi29018 expresses phi29-F136CA376CM7RV50AM96TG196DE220KQ496P gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50AM96TG196DE220KQ496P. The amino acid sequence of the mutant F136CA376CM7RV50AM96TG196DE220KQ496P is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, M at 96 th position is replaced by T, G at 196 th position is replaced by D, E at 220 th position is replaced by K, Q at 496 th position is replaced by P, and other amino acid residues are unchanged.

Plasmid pZJphi29019 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by the phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511E gene, the other nucleotides remaining unchanged. The modified phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511E gene sequence is characterized in that the optimized phi29 wild-type gene sequence is replaced by TTT (F codon) at 406 th to 408 th positions, TGC (C codon) is replaced by GCG (A codon) at 1126 th to 1128 th positions, CGT (R codon) is replaced by ATG (M codon) at 19 th to 21 th positions, GCG (A codon) is replaced by GTG (V codon) at 148 th to 150 th positions, ATG (M codon) at 586 th to 288 th positions is replaced by ACC (T codon), GGT (G codon) at 586 th to 588 th positions is replaced by GAC (D codon) at 658 th to 660 th positions, GAA (E codon) at 1486 th to 8 th positions is replaced by AAA (K codon), CAG (Q codon) at 148 th to 21 th positions is replaced by CCG (P codon), and AAK (K) at 148 th to 150 th positions is replaced by GAA (E) at rest. Plasmid pZJphi29019 expresses phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511E gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50AM96TG196DE220KQ496PK511E. The amino acid sequence of the mutant F136CA376CM7 AM96TG196DE220KQ496PK511E is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, M at 96 th position is replaced by T, G at 196 th position is replaced by D, E at 220 th position is replaced by K, Q at 496 th position is replaced by P, K at 511 th position is replaced by E, and other amino acid residues are unchanged.

Plasmid pZJphi29020 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced by the phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L gene, the other nucleotides remaining unchanged. The modified phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L gene sequence is characterized in that the optimized TTT (F codon) at 406 th to 408 th positions of the phi29 wild type gene sequence is replaced by TGC (C codon), the GCG (A codon) at 1126 th to 1128 th positions is replaced by TGC (C codon), the ATG (M codon) at 19 th to 21 th positions is replaced by CGT (R codon), the GTG (V codon) at 148 th to 150 th positions is replaced by GCG (A codon), the ATG (M codon) at 286 th to 288 th positions is replaced by ACC (T codon), the GGT (G codon) at 586 th to 588 th positions is replaced by GAC (D codon) at 658 th to 660 th positions of GAA (E codon), the CAG (Q codon) at 1486 th to 1488 th positions is replaced by CCG (P codon), the G (K) at 148 th to 1533 th positions is replaced by GAC (G codon) at 1575 th to 15 th nucleotide, and the other nucleotide substitutions (TTL) are not changed. Plasmid pZJphi29020 expresses phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L gene, and the expression product is phi29DNA polymerase mutant F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L. The amino acid sequence of the mutant F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L is that F at 136 th position is replaced by C, A at 376 th position is replaced by C, M at 7 th position is replaced by R, V at 50 th position is replaced by A, M at 96 th position is replaced by T, G at 196 th position is replaced by D, E at 220 th position is replaced by K, Q at 496 th position is replaced by P, K at 511 th position is replaced by E, F at 525 th position is replaced by L, and other amino acid residues are unchanged.

Plasmid pZJphi29021 differs from pET-21c (+) -phi29 wild-type plasmid only in that the optimized phi29 wild-type gene contained in pET-21c (+) -phi29 wild-type is replaced with a DNA fragment containing His-GB1 gene and phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L gene. The nucleotide sequence of the replaced DNA fragment is shown as SEQ ID No.2, wherein, the 1 st to 75 th positions are His and the nucleotide sequence of the added connecting gene, the 76 th to 237 th positions are the nucleotide sequence of the GB1 gene, the 238 th to 324 th positions are the nucleotide sequence of the added connecting gene, and the 325 th to 2046 th positions are phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L gene nucleotide sequence. Plasmid pZJphi29021 expresses phi29-F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L gene and His-GB1 gene, and the expression products are fusion proteins comprising GB1 protein and phi29DNA polymerase mutant F136CA376CM7RV50AM96TG196DE220KQ496PK511EF525L, the amino acid sequence of the expression products is shown as SEQ ID No.3, wherein, the 1 st to 25 th positions are His and the amino acid sequence of the added connecting gene, the 26 th to 79 th positions are the amino acid sequence of the GB1 protein, the 80 th to 108 th positions are the amino acid sequence of the added connecting gene, and the 109 th to 682 th positions are mutant F136CA376CM7RV50AM96TG196DE220KQ496 511EF525L amino acid sequence.

The specific construction method of the plasmid is as follows:

1) The wild type gene phi29 is synthesized by performing escherichia coli codon optimization and then inserted into a pET-21c (+) vector by Shanghai engineering and bioengineering company to construct a plasmid pET-21c (+) -phi29 wild type. The plasmid pET-21C (+) -phi29 wild type is extracted, and the extracted pET-21C (+) -phi29 wild type plasmid is used as a template, and KOD Plus Neo DNA polymerase (TOYOBO) is used for carrying out PCR by using the primers K6C-A55C-F1 and K6C-A55C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29001a with a mutation in position. Then, the plasmid pZJphi29001a was used as a template, and the primers K6C-A55C-F2 and K6C-A55C-R2 were used to amplify the plasmid pZJphi29001a by PCR using KOD Plus Neo DNA polymerase (TOYOBO), and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29001.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers V23C-L43C-F1 and V23C-L43C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29002a with a mutation in position. Then, the whole plasmid of pZJphi29002a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers V23C-L43C-F2 and V23C-L43C-R2 using the plasmid pZJphi29002a as a template, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29002.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR, primers Y58C-S121C-F1 and Y58C-S121C-R1 are used for PCR, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29003a with a mutation in position. Then, the whole plasmid of pZJphi29003a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers Y58C-S121C-F2 and Y58C-S121C-R2 using the plasmid pZJphi29003a as a template, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29003.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR, primers E74C-L405C-F1 and E74C-L405C-R1 are used for PCR, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29004a with a mutation site. Then, the whole plasmid of pZJphi29004a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers E74C-L405C-F2 and E74C-L405C-R2 using the plasmid pZJphi29004a as a template, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29004.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers F136C-A376C-F1 and F136C-A376C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29005a with a mutation site. Then, the whole plasmid of pZJphi29005a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers F136C-A376C-F2 and F136C-A376C-R2, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29005.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers S193C-S387C-F1 and S193C-S387C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29006a with a mutation at a position. Then, the whole plasmid pZJphi29006a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers S193C-S387C-F2 and S193C-S387C-R2 using the plasmid pZJphi29006a as a template, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29006.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers L252C-D457C-F1 and L252C-D457C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29007a with a mutation in position. Then, the whole plasmid of pZJphi29007a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers L252C-D457C-F2 and L252C-D457C-R2 using the plasmid pZJphi29007a as a template, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29007.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR, primers Y253C-Y389C-F1 and Y253C-Y389C-R1 are used for carrying out enzyme digestion treatment on an amplified product by using Dpn I to obtain a plasmid pZJphi29008a with a mutation at a position. Then, the whole plasmid of pZJphi29008a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers Y253C-Y389C-F2 and Y253C-Y389C-R2, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29008.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers M257C-F359C-F1 and M257C-F359C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29009a with a mutation site. Then, the whole plasmid of pZJphi29009a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers M257C-F359C-F2 and M257C-F359C-R2, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29009.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers Y280C-K351C-F1 and Y280C-K351C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29010a with a mutation in position. Then, the whole plasmid of pZJphi29010a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers Y280C-K351C-F2 and Y280C-K351C-R2, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29010.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers Y297C-H338C-F1 and Y297C-H338C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29011a with a mutation at a position. Then, the whole plasmid of pZJphi29011a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO) and primers Y297C-H338C-F2 and Y297C-H338C-R2, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29011.

The extracted pET-21C (+) -phi29 wild type plasmid is used as a template, KOD Plus Neo DNA polymerase (TOYOBO) is used for PCR by using primers V330C-A434C-F1 and V330C-A434C-R1, and the amplified product is subjected to Dpn I enzyme digestion to obtain a plasmid pZJphi29012a with a mutation site. Then, the whole plasmid of pZJphi29012a was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers V330C-A434C-F2 and V330C-A434C-R2, and the plasmid was digested with Dpn I to obtain two site-mutated plasmids designated pZJphi29012.

The specific operation steps of mutant plasmid acquisition are as follows:

(1) PCR system for amplifying whole plasmid with pET-21c (+) -phi29 wild type plasmid as template:

PCR program

(2) And (3) carrying out enzyme digestion on the amplified product by using Dpn I, and recovering the product to obtain the plasmid containing the point mutation.

Cleavage system of amplified products:

2) Extracting a plasmid pZJphi29005, carrying out PCR (polymerase chain reaction) by using KOD Plus Neo DNA polymerase (TOYOBO) and primers M7R-F and M7R-R by taking the extracted pZJphi29005 plasmid as a template, and carrying out enzyme digestion on an amplified product by using Dpn I to obtain a plasmid pZJphi29013 with 3 site mutations; using plasmid pZJphi29013 as a template, carrying out PCR amplification on the whole plasmid pZJphi29013 by using KOD Plus Neo DNA polymerase (TOYOBO) and primers V50A-F and V50A-R, and carrying out enzyme digestion treatment by using Dpn I to obtain 4 site-mutated plasmids which are named pZJphi29014; using plasmid pZJphi29014 as a template, carrying out PCR amplification on the whole plasmid pZJphi29014 by using KOD Plus Neo DNA polymerase (TOYOBO) and primers M96T-F and M96T-R, and carrying out enzyme digestion treatment by using Dpn I to obtain a plasmid with 5 site mutations, which is named pZJphi29015; using plasmid pZJphi29015 as a template, carrying out PCR amplification on the whole plasmid pZJphi29015 by using KOD Plus Neo DNA polymerase (TOYOBO) and primers G196D-F and G196D-R, and carrying out enzyme digestion treatment by using Dpn I to obtain a plasmid with 6 site mutations, which is named pZJphi29016; using plasmid pZJphi29016 as a template, carrying out PCR amplification on the whole plasmid pZJphi29016 by using KOD Plus Neo DNA polymerase (TOYOBO) and primers E220K-F and E220K-R, and carrying out enzyme digestion treatment by using Dpn I to obtain 7 site-mutated plasmid named pZJphi29017; using plasmid pZJphi29017 as a template, carrying out PCR amplification on the whole plasmid pZJphi29017 by using KOD Plus Neo DNA polymerase (TOYOBO) and primers Q496P-F and Q496P-R, and carrying out enzyme digestion treatment by using Dpn I to obtain 8 site-mutated plasmid named pZJphi29018; using plasmid pZJphi29018 as a template, carrying out PCR amplification on the whole plasmid pZJphi29018 by using KOD Plus Neo DNA polymerase (TOYOBO) and primers K511E-F and K511E-R, and carrying out enzyme digestion treatment by using Dpn I to obtain 9 site-mutated plasmid which is named pZJphi29019; the plasmid pZJphi29019 was used as a template, and the whole plasmid of pZJphi29019 was amplified by PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers F525L-F and F525L-R, and the plasmid was digested with Dpn I to obtain 10 site-mutated plasmid designated pZJphi29020.

The specific operation steps of mutant plasmid acquisition are as follows:

(1) PCR system for amplifying whole plasmid with corresponding plasmid as template:

PCR program

Cleavage system of amplified products:

3) The extracted pZJphi29020 plasmid is used as a template, Q5 High-Fidelity DNA polymerase is used for PCR amplification of the skeleton by using primers 036A-F and 036A-R, and the amplified product is digested by DpnI after gel cutting and recovery. Designing oligonucleotides (036A-1-8) which are overlapped with His-GB1, diluting the overlapped oligonucleotides by a certain multiple, mixing, performing SOE PCR by using PrimeSTAR Max DNA polymerase, performing two stages in one round of PCR, wherein the first stage is 25 cycles, and annealing and extending the oligonucleotides to form a long fragment; in the second stage, 35 cycles, primers 036A-1 and 036A-8 were added for extension amplification. And (3) cutting and recovering the PCR product to obtain the His-GB1 gene fragment. The digested skeleton is subjected to seamless cloning connection with a target gene, so that a plasmid containing 10 site mutation phi29 genes and GB1 genes is obtained and is named pZJphi29021 (the structure is shown as figure 1).

The specific operation steps are as follows:

(1) PCR System for amplifying the full plasmid Using pZJphi29020 plasmid as template:

PCR program

(2) DpnI digestive system (10. Mu.l):

(3) the specific operation steps of the His-GB1 target gene acquisition are as follows:

PCR system for gene synthesis:

PCR program

Second round PCR

PCR program

And (5) after the second round of PCR product gel cutting recovery, obtaining the His-GB1 gene fragment.

(4) And (3) performing seamless cloning connection on the digested plasmid skeleton and the target gene.

The digested skeleton is subjected to seamless cloning connection with a target gene, so that a plasmid containing 10 site mutation phi29 genes and GB1 genes is obtained and is named pZJphi29021.

2. Transferring the constructed vector into an expression vector escherichia coli BL 21:

the site-directed mutated 20 plasmids were transformed into E.coli BL21 cells of the host strain, respectively.

Specific procedure for constructing various phi29 DNA polymerase mutant expression strains:

the pET-21c (+) -phi29 wild type plasmid is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: phi29 wild type;

The plasmid pZJphi29001 containing K6C site and A56C site mutation is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/1, naCl 10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29001;

the plasmid pZJphi29002 containing the V23C site and the L43C site mutation is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29002;

the plasmid pZJphi29003 containing the mutation at the Y58C position and the S121C position is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29003;

The plasmid pZJphi29004 containing E74C site and L405C site mutations is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance and the function of phi29DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the resulting positive strain transformants were identified by sequencing as: EZJphi29004;

the plasmid pZJphi29005 containing F136C site and A376C site mutations is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29005;

the plasmid pZJphi29006 containing S193C and S387C mutations is transformed into BL21 escherichia coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/1, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29006;

The plasmid pZJphi29007 containing the L252C site and the D457C site mutation is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29007;

the plasmid pZJphi29008 containing the mutation at the Y253C position and the Y389C position is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/1, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29008;

the plasmid pZJphi29009 containing M257C and F359C mutations is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29009;

The plasmid pZJphi29010 containing Y280C site and K351C site mutations is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/1, naCl10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29010;

plasmid pZJphi29011 containing mutations at Y297C and H338C is transformed into BL21 E.coli, so that the strain recovers the function of Amp resistance and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29011;

the plasmid pZJphi29012 containing the mutations at the V330C position and the A434C position is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29012;

The plasmid pZJphi29013 containing F136C site, A376C site and M7R site mutations is transformed into BL21 escherichia coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29013;

the plasmid pZJphi29014 containing F136C site, A376C site, M7R site and V50A site mutations is transformed into BL21 escherichia coli, so that the strain recovers the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29014;

plasmid pZJphi29015 containing F136C, A376C, M7R, V50A and M96T mutations is transformed into BL21 E.coli, so that the strain recovers the function of Amp resistance and the function of phi29 DNA polymerase is expressed in a new way. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29015;

Plasmid pZJphi29016 containing F136C, A376C, M7R, V50A, M96T and G196D mutations is transformed into BL21 E.coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29016;

plasmid pZJphi29017 containing F136C, A376C, M7R, V50A, M96T, G196D and E220K mutations is transformed into BL21 E.coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29017;

plasmid pZJphi29018 containing F136C, A376C, M7R, V50A, M96T, G196D, E220K and Q496P mutations is transformed into BL21 E.coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/1), and the positive strains obtained were named as follows by sequencing verification: EZJphi29018;

Plasmid pZJphi29019 containing F136C, A376C, M7R, V50A, M96T, G196D, E220K, Q496P and K511E mutations is transformed into BL21 E.coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29019;

plasmid pZJphi29020 containing F136C, A376C, M7R, V50A, M96T, G196D, E220K, Q496P, K511E and F525L mutations is transformed into BL21 E.coli, so that the strain is recovered to have the function of Amp resistance, and the function of phi29 DNA polymerase is newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/1, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29020;

the plasmid pZJphi29021 containing F136C, A376C, M7R, V50A, M96T, G196D, E220K, Q496P, K511E and F525L and having a GB1 gene sequence is transformed into BL21 escherichia coli, so that the strain is recovered to have the function of Amp resistance, and the functions of phi29 DNA polymerase and GB1 fusion protein are newly expressed. Positive clones were selected on medium containing Amp resistance (formulation: tryptone 10g/l, yeast extract 5g/l, naCl 10 g/l), and the positive strains obtained were named as follows by sequencing verification: EZJphi29021.

The specific experimental method is as follows.

1) E.coli chemical conversion step:

(1) taking out a tube (100 ul) of competent bacteria from a freezer with ultralow temperature of-70 ℃, immediately heating with fingers to melt, then inserting the melted bacteria on ice, and carrying out ice bath for 5-10 min.

(2) Adding 5ul of the plasmid mixture (DNA content not more than 100 ng), gently shaking, and standing on ice for 20min.

(3) Slightly shaking, then inserting into a water bath at 42 ℃ for 45s for heat shock, then quickly putting back into ice, and standing for 3-5 min.

(4) Add 500ul LB medium (without antibiotics) into each tube in ultra clean bench, mix gently, then fix to spring rack of shaking table and shake for 1h at 37deg.C.

(5) And (3) taking 100-300ul of the transformation mixed solution in an ultra-clean workbench, respectively dripping the solution into solid LB flat plate culture dishes containing proper antibiotics, and uniformly coating the solid LB flat plate culture dishes by using a glass coating rod burnt by an alcohol lamp.

(6) Marking the coated culture dish, placing the culture dish in a constant temperature incubator at 37 ℃ for 30-60 min until the surface liquid permeates into the culture medium, and placing the culture dish in the constant temperature incubator at 37 ℃ for overnight after inversion.

(7) Picking up the clone on the plate, culturing in liquid LB to obtain monoclonal, and carrying out first generation sequencing verification on the monoclonal to obtain the strain containing the correct gene sequence (namely containing the corresponding target gene sequence), namely the positive strain.

Example 2 comparison of the Single cell genomic amplification Capacity of different phi29 DNA polymerase mutants

This example was used to compare the level of protease activity expressed by different strains of phi29 DNA polymerase mutants. The result shows that the activity stability of the protein phi29 DNA polymerase mutant expressed by the EZJphi29020 strain is highest.

1. Strains were resuscitated on LB medium plates. Control strain: phi29 wild type prepared in example 1. Experiment strain: EZJphi29001, EZJphi29002, EZJphi29003, EZJphi29004, EZJphi29005, EZJphi29006, EZJphi29007, EZJphi29008, EZJphi29009, EZJphi29010, EZJphi29011, EZJphi29012, EZJphi29020 prepared in example 1. The cells were incubated overnight at 37 ℃.

2. The individual clones were picked up and inoculated into 5ml of liquid LB medium. Culturing at 37℃at 250rpm overnight.

3. Prepare 42 bottles of 200mL LB medium and split into 1L conical flasks. The formula comprises the following components: 10g/l tryptone, 5g/l yeast extract and 10g/l NaCl. Sterilizing for standby.

4. The overnight culture in the step 2 was inoculated into 200mL of LB medium, and the volume ratio of the overnight culture to the LB medium was 1:100, 37℃and 250rpm for cultivation. Each strain was repeated 3 times.

5. And 4, culturing until the OD600 = 0.8 or so, adding IPTG with the final concentration of 1mM for induced expression, and carrying out induced expression at 16 ℃ for 21h, collecting bacteria, crushing the bacteria, and purifying the protein.

The protein purification steps are as follows:

1) All reagents used below were pre-chilled at 4 degrees and the following experimental runs were guaranteed to be performed on ice.

2) The protein-expressing strain (i.e., the strain cultured in step 4 to induce 21 h) taken out of the-80℃refrigerator was washed once with 20ml of lysies buffer (20 mM Tris-HCl (pH 7.6), 300mM KCl and 5mM imidozole (imidazole)), centrifuged at 5000 Xg at 4℃for 5 minutes to obtain precipitated cells, and the cells were suspended in 20ml of lysies buffer.

3) Then, step 2) cells were lysed by ultrasound in an ice bath with a 1 second pulse at 30% intensity (intensity 30%; crushing for 1 second and stopping for 5 seconds; run for 45 minutes) to obtain lysate.

4) After completion of the disruption, the lysate of step 3) was centrifuged at 10000×g at 4℃for 30 minutes, and cell debris was precipitated at 4℃to obtain a supernatant for protein purification.

5) To the affinity column, 500. Mu.L of Ni-NTA matrix, 10mL ddH2O,10mL Lysis buffer, step 4) protein supernatant, 10mL Wash buffer I (20 mM Tris-HCl (pH 7.6), 300mM KCl and20mM imidazole), 10mL Wash buffer II (20 mM Tris-HCl (pH 7.6), 300mM KCl and 40mM imidazole), 3mL purification eluate (20 mM Tris-HCl (pH 7.6), 300mM KCl and 250mM imidazole) were added.

6) Finally, the eluted protein of step 5) was collected with a 1.5mL centrifuge tube (10 tubes were collected in total, 200. Mu.L per tube).

7) mu.L of the protein of step 6) was mixed with 2X protein loading buffer per tube, and after cooling to room temperature for 10 minutes, it was centrifuged at 14000X rpm for 4 minutes at room temperature, followed by detection with 12% SDS-PAGE gel.

8) The remaining step 6) protein samples were stored at 4 degrees for later use.

9) Dialysis bag buffer I (2%NaHCO3,1mM EDTA-Na2.2H2O) and dialysis bag buffer II (1 mM EDTA-Na2.2H2O) were boiled.

10 Putting the dialysis bag into a boiled dialysis bag buffer I, boiling for 10 minutes, and thoroughly washing with ultrapure water; then put into a dialysis bag buffer II, boiled for 10 minutes and thoroughly washed by ultrapure water.

11 Placing the dialysis bag treated in the step 10) at 4 ℃ for standby.

12 After running out of step 7) 12% SDS-PAGE gel, several tubes with defined protein are pooled and added to step 11) dialysis bags for storage, the openings are closed with clamps (four-degree chromatography cabinet cooling is opened in advance).

13 The dialysis bag with the port closed in step 12) was placed in a large beaker containing the rotor and approximately 500mL storage buffer (40 mM Tris-HCl (pH 7.6), 50mM KCl,5mM (NH 4) 2SO4, 10mM MgCl2).

14 Place step 13) beaker on a magnetic stirrer in a 4 degree chromatography cabinet.

15 Standing the large beaker of step 14) overnight.

16 The next morning and noon, step 15) store buffer in the beaker is changed twice. A total of 3 times of dialysis, each for 4 hours.

17 Recovering the dialyzed protein and preserving at 4 ℃.

Protein concentration determination:

1) The sample (i.e., the recovered protein of the above protein purification step 17) was diluted with distilled water at appropriate times (10 times, 20 times, 40 times).

2) A1.5 mL centrifuge tube was added to each solution in the proportions set forth in Table 1 below, wherein tube 0 was a blank.

TABLE 1 BSA Standard protein solution Components

3) 30 1.5mL centrifuge tubes were taken and divided into standard and sample groups.

Standard group: 18 1.5mL centrifuge tubes were set up for each standard with 2 replicates, and each tube was added with 100. Mu.L of the corresponding concentration BSA standard protein solution prepared in step 2), designated as tube numbers 0-9 according to Table 1.

Sample group: 12 1.5mL centrifuge tubes are divided into 3 repeated groups, the same repeated centrifuge tube in different groups has the same serial number, and 100 mu L of sample diluents with different concentrations diluted in the step 1) are respectively added into the two tubes with different serial numbers, and the sample dilutions are respectively 0 times, 10 times, 20 times and 40 times.

4) Step 3) each tube was added with 1ml of a radford working solution (Shanghai Bioengineering Co., C503031) and mixed rapidly.

5) After the centrifuge tube treated in the step 4) was reacted for 10 minutes at room temperature of 25 degrees, the A595 value of each tube was measured on a spectrophotometer with the tube No. 0 as a blank.

6) Standard curves were plotted in Microsoft Excel software with the mean value of each tube a595 of the standard group on the ordinate and the corresponding protein concentration on the abscissa.

7) According to the average value of the A595 values of two identical sample dilutions, the protein concentration of the diluted sample is calculated on a standard curve, the final sample protein concentration is calculated by selecting a sample with proper dilution, and the original sample protein concentration is calculated by dilution multiple.

6. Detecting the protease activity obtained in the step 5.

Phi29 DNA polymerase activity assay:

1) Standard curves for fluorescence intensity-enzyme activity units were established with commercial kits (Thermo):

the system was as follows (50 ul):

10×phi29 reaction Buffer：5μL

random primer (10 uM): 10 mu L

HindIII-digested pellet DNA (0.1 mg/ml): 5 mu L

Pre-denatured at 95 ℃ for 3min, and cooled on ice.

dNTP(2mM)：5μL

10mg/ml BSA：0.5μL

Gradient diluted phi29 DNA polymerase: 1 mu L

1x SYBR Green I：23.5μL

30 ℃ C:: 4 hours

65 ℃ C: and (5) inactivating for 10 minutes.

2) After the reaction, excitation was performed at 480nm using an enzyme-labeled instrument, and an absorption peak at 520m was measured.

3) The assay group enzyme (i.e., the recovered protein of the above protein purification step 17) was reacted according to the above system (1. Mu.L enzyme), and the fluorescence intensity was measured and substituted into the standard curve to define the activity unit (U/. Mu.L).

7. Genome amplification was performed using the protein obtained in step 5.

The specific experimental method comprises the following steps:

1) Preparing sufficient lysate (Lysis bufferA) for single cell genome amplification reaction procedure according to table 2;

TABLE 2 composition ratio of lysate (Lysis bufferA)

Component (A)	Measuring amount
		Lysis buffer A1(0.1mM KOH、1mM EDTA)	33μl
Lysis buffer A2(1M DTT)	3μl
		Total amount of	36μl

2) Mu.l of E.coli DH5a cell (Shanghai Biotechnology Co.) material (said cell material utilizing H ₂ O resuspended, individual cells were isolated directly) and placed in a microcentrifuge tube, 1. Mu.l of Lysis buffer A was added, thoroughly mixed with shaking, and centrifuged briefly. Incubating at 65 ℃ for 10-15min. After the addition of 1 mu l Neutralization buffer B (60 mM KH2PO4 and 5mM K2 HP)O4). Shaking and mixing thoroughly, and centrifuging briefly to obtain denatured DNA with concentration of fg grade, and storing on ice.

3) Placing the enzyme of the experimental group (i.e. the recovered protein of the protein purification step 17) on ice for later use; before other reagents are used, the reagents are dissolved at room temperature, vibrated and centrifuged and placed on ice for standby; NF water is placed at room temperature for standby; the SCC-reaction buffer C solution consisted of 10% volume of 10 XPhi 29 buffer (300 mM Tris-HCl,300mM KCl,100mM (NH 4) 2SO4, 100mM MgCl2), water, 7.5% volume of DMSO,10mM dNTP, N6 primers (Integrated DNA Technologies (IDT)) at a total primer concentration of 50. Mu.M, and 1M DTT. An amplification mixture was prepared according to table 3. Mixing water (NF water), SCC-reaction buffer C1, shaking, mixing, centrifuging, and adding enzyme of experimental group to obtain amplification mixed solution.

TABLE 3 preparation of amplification mixture

4) In each reaction, 10. Mu.l of the reaction mixture prepared in step 3) was added to 3. Mu.l of the denatured DNA obtained in step 2). Incubate at 30℃for 8 hours. The polymerase activity was inactivated at 65℃for 10min to obtain amplified DNA. The amplified DNA was subjected to agarose gel electrophoresis.

Diluting the amplified DNA by 20 times, taking 1 μl of the amplified DNA as a template, carrying out PCR amplification 16S detection by using primers 27F and 1492R, and carrying out agarose gel electrophoresis on the amplified product, wherein the template of the amplified strip is the MDA product which is successfully amplified. The successfully amplified MDA product was purified and second-generation sequenced. The negative control is the template of empty droplets, and the positive control is the template of E.coli DH5a cell ng grade genome.

27F：AGAGTTTGATCCTGGCTCAG

1492R：TACGGYTACCTTGTTACGACTT

Results of enzyme activity detection EZJphi29 wild type (wild type), EZJphi29001 (E1), EZJphi29002 (E2), EZJphi29004 (E4), EZJphi29005 (E5), EZJphi29008 (E8), EZJphi29010 (E10), EZJphi29020 (E20) expressed protease activities of 0.82U/μl, 2.72U/μl, 3.06U/μl, 3.04U/μl, 3.7U/μl, 0.65U/μl, 3.15U/μl, 5.99U/μl, respectively (FIG. 2C). The proteins expressed by EZJphi29003, EZJphi29006, EZJphi29007, EZJphi29009, EZJphi29011 and EZJphi29012 were not detected by the method for detecting enzyme activity described above.

The results are shown in FIG. 2A and FIG. 2B, wherein FIG. 2A is a graph of the amplification result of the amplification genome of the phi29 DNA polymerase mutant without activity, FIG. 2B is a graph of the amplification result of the amplification genome of the phi29 DNA polymerase mutant with activity, MDA is the detection result of agarose gel electrophoresis of the amplified DNA, and 27F/1492R is the detection result of 16S amplification. As can be seen from FIG. 2A, the proteins purified from the strains EZJphi29003, EZJphi29006, EZJphi29007, EZJphi29009, EZJphi29011 and EZJphi29012 did not amplify the bands and lost the enzyme activity as DNA polymerase amplified genomes. As can be seen from FIG. 2B, the proteins purified from the strains EZJphi29001, EZJphi29002, EZJphi29004, EZJphi29005, EZJphi29008, EZJphi29010 and EZJphi29020 were amplified as DNA polymerase amplified genomes, and were all amplified to have enzyme activities, wherein the mutant expressed by EZJphi29005 and the phi29 mutant expressed by EZJphi29020 were amplified as DNA polymerase for 16S amplification, the amplification success rate was 100%, and genome amplification at single cell level was completed. Second-generation sequencing is carried out on MDA products amplified by a mutant expressed by the strain EZJphi29005 and a phi29 mutant expressed by the strain EZJphi29020, and from the aspect of genome sequencing, the phi29 DNA polymerase mutant expressed by the strain EZJphi29005 can realize 74% of genome amplification coverage, and the phi29 DNA polymerase mutant expressed by the strain EZJphi29020 can realize 90% of genome amplification coverage.

Example 3 fusion expression of GB1 protein to increase expression level of phi29 DNA polymerase mutant

This example was used to verify the effect of fusion expression of the GB1 protein on the expression level of phi29 DNA polymerase mutants. The results showed that the amplification ability of the protease with increased expression level was still maintained, and at the same time, the soluble expression level was increased by 2.7 times.

1. Strains were resuscitated on LB medium plates. Control strain: EZJphi29020 prepared in example 1. Experiment strain: EZJphi29021 prepared in example 1. The culture was carried out at 37℃for 1 day.

2. The individual clones were picked up and inoculated into 5ml of liquid LB medium. 37℃at 250rpm overnight.

3. 6 bottles of 50mL LB medium were prepared and dispensed into 250mL Erlenmeyer flasks. The formula comprises the following components: 10g/l tryptone, 5g/l yeast extract and 10g/l NaCl. Sterilizing for standby.

5. Step 4, when the culture was carried out until the OD 600=0.8 or so, IPTG was added at a final concentration of 1mM to induce expression, and the induction expression was carried out at 16℃for 21 hours, followed by harvesting and disruption of the cells, and the purified protein was obtained in the same manner as in step 5 of example 2.

6. The enzyme activity and yield of the purified protein of step 5 were examined. The method for detecting the enzyme activity was the same as in step 6 of example 2. The method for detecting the yield was the same as the protein concentration measurement in step 5 in example 2.

As shown in FIG. 3, EZJphi29021 can express 9.49mg of enzyme by using 1L of fermentation broth, while EZJphi29020 can only express 3.52mg of enzyme by using 1L of fermentation broth; the protease activity expressed by EZJphi29020 is 5.99U/. Mu.l, and the protease activity expressed by EZJphi29021 is 6.19U/. Mu.l. As can be seen from FIG. 3, EZJphi29021 containing the GB1 fusion expression protein increased the expression level of the final soluble protein by 2.7 times as compared with EZJphi29020 containing no GB1 fusion expression protein.

The primers and DNA fragment sequences used in the above examples are as follows:

TABLE 4 primers and DNA fragment sequences

/>

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> Qingdao bioenergy and Process institute of China academy of sciences

<120> single cell genome amplifying enzyme mutant and application thereof

<130> 211963

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1728

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgaaacaca tgccgcgtaa aatgtactct tgcgatttcg aaaccaccac caaagtggaa 60

gattgccgtg tttgggcgta cggctacatg aacatcgaag atcacagcga atacaaaatc 120

ggcaacagcc tggacgaatt catggcctgg gtgctgaaag ttcaggcgga cctgtacttc 180

cacaacctga aattcgatgg cgcgttcatc atcaactggc tggaacgtaa cggctttaaa 240

tggagcgcgg acggtctgcc gaacacctac aacaccatca tttcccgcat gggtcagtgg 300

tatatgattg atatctgcct gggctacaaa ggtaaacgta aaatccacac cgttatctac 360

gacagcctga aaaaactgcc gttcccggtt aaaaagatcg cgaaagattt taaactgacc 420

gttctgaaag gcgatatcga ttaccacaaa gaacgcccgg ttggctacaa aatcaccccg 480

gaagaatatg cttatatcaa aaacgacatc cagatcatcg ctgaagcgct gctgatccag 540

ttcaaacagg gcctggatcg tatgaccgcg ggcagcgata gcctgaaagg tttcaaagac 600

atcatcacca ccaaaaaatt taaaaaagtt ttcccgaccc tgtccctggg tctggacaaa 660

gaagtgcgct atgcgtatcg cggtggtttc acctggctga atgatcgttt caaagaaaag 720

gaaatcggcg aaggtatggt gttcgacgtg aactccctgt acccggcgca gatgtactct 780

cgtctgctgc cgtatggcga accgatcgtg ttcgaaggca agtacgtttg ggacgaagac 840

tacccactgc acatccagca catccgttgc gaattcgaac tgaaagaggg ttacatcccg 900

accatccaaa tcaaacgttc tcgcttctat aaaggcaacg aatacctgaa aagctccggt 960

ggtgaaatcg ctgatctgtg gctgtccaac gtggacctgg aactgatgaa agaacattat 1020

gacctgtaca acgttgaata catctccggc ctgaaattca aagccaccac gggcctgttc 1080

aaagacttca tcgataaatg gacctacatc aaaaccacta gcgaaggtgc gattaaacag 1140

ctggcgaaac tgatgctgaa ctccctgtac ggtaaatttg cttctaaccc ggacgtgacc 1200

ggcaaagttc cgtacctgaa agaaaacggc gcactgggtt tccgcctggg cgaagaagaa 1260

accaaagacc cagtgtacac cccgatgggc gttttcatca ccgcatgggc tcgttacact 1320

accatcaccg cggctcaggc gtgctatgat cgtattatct actgcgatac cgactctatc 1380

cacctgaccg gcactgaaat cccggacgtt atcaaagata tcgttgaccc taaaaaactg 1440

ggttattggg cgcacgaatc taccttcaaa cgtgcgaaat atctgcgtca gaaaacctac 1500

atccaggaca tctatatgaa agaagttgat ggcaagctgg tggaaggtag cccggatgat 1560

tacaccgaca tcaaatttag cgtgaaatgc gccggcatga ccgacaaaat caaaaaagaa 1620

gtaacctttg aaaacttcaa agtgggcttt agccgtaaaa tgaaaccgaa accggttcag 1680

gttccgggcg gtgttgttct ggttgatgac accttcacca ttaaataa 1728

<210> 2

<211> 2046

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgggcagca gccatcatca tcatcatcac caccaccatc atgccgtggg cggatccggt 60

ggttcaggcg gtagttataa attgatcctg aacggcaaaa ctttgaaggg cgaaacaaca 120

acagaagctg ttgacgctgc taccgctgaa aaagtattta aacagtatgc aaacgacaac 180

ggtgttgatg gagaatggac atacgatgac gcaaccaaaa ccttcactgt cacagaaggt 240

ggtgggagcg gaggcggtgg cagcggtggc ggcggtagtg gcggtggcgg ttcgggcggc 300

ggtggttcgg gtggcggagg cagcaaacac atgccgcgta aacgttactc ttgcgatttc 360

gaaaccacca ccaaagtgga agattgccgt gtttgggcgt acggctacat gaacatcgaa 420

gatcacagcg aatacaaaat cggcaacagc ctggacgaat tcatggcctg ggcgctgaaa 480

gttcaggcgg acctgtactt ccacaacctg aaattcgatg gcgcgttcat catcaactgg 540

ctggaacgta acggctttaa atggagcgcg gacggtctgc cgaacaccta caacaccatc 600

atttcccgca ccggtcagtg gtatatgatt gatatctgcc tgggctacaa aggtaaacgt 660

aaaatccaca ccgttatcta cgacagcctg aaaaaactgc cgttcccggt taaaaagatc 720

gcgaaagatt gcaaactgac cgttctgaaa ggcgatatcg attaccacaa agaacgcccg 780

gttggctaca aaatcacccc ggaagaatat gcttatatca aaaacgacat ccagatcatc 840

gctgaagcgc tgctgatcca gttcaaacag ggcctggatc gtatgaccgc gggcagcgat 900

agcctgaaag acttcaaaga catcatcacc accaaaaaat ttaaaaaagt tttcccgacc 960

ctgtccctgg gtctggacaa aaaagtgcgc tatgcgtatc gcggtggttt cacctggctg 1020

aatgatcgtt tcaaagaaaa ggaaatcggc gaaggtatgg tgttcgacgt gaactccctg 1080

tacccggcgc agatgtactc tcgtctgctg ccgtatggcg aaccgatcgt gttcgaaggc 1140

aagtacgttt gggacgaaga ctacccactg cacatccagc acatccgttg cgaattcgaa 1200

ctgaaagagg gttacatccc gaccatccaa atcaaacgtt ctcgcttcta taaaggcaac 1260

gaatacctga aaagctccgg tggtgaaatc gctgatctgt ggctgtccaa cgtggacctg 1320

gaactgatga aagaacatta tgacctgtac aacgttgaat acatctccgg cctgaaattc 1380

aaagccacca cgggcctgtt caaagacttc atcgataaat ggacctacat caaaaccact 1440

agcgaaggtt gcattaaaca gctggcgaaa ctgatgctga actccctgta cggtaaattt 1500

gcttctaacc cggacgtgac cggcaaagtt ccgtacctga aagaaaacgg cgcactgggt 1560

ttccgcctgg gcgaagaaga aaccaaagac ccagtgtaca ccccgatggg cgttttcatc 1620

accgcatggg ctcgttacac taccatcacc gcggctcagg cgtgctatga tcgtattatc 1680

tactgcgata ccgactctat ccacctgacc ggcactgaaa tcccggacgt tatcaaagat 1740

atcgttgacc ctaaaaaact gggttattgg gcgcacgaat ctaccttcaa acgtgcgaaa 1800

tatctgcgtc cgaaaaccta catccaggac atctatatga aagaagttga tggcgaactg 1860

gtggaaggta gcccggatga ttacaccgac atcaaactga gcgtgaaatg cgccggcatg 1920

accgacaaaa tcaaaaaaga agtaaccttt gaaaacttca aagtgggctt tagccgtaaa 1980

atgaaaccga aaccggttca ggttccgggc ggtgttgttc tggttgatga caccttcacc 2040

attaaa 2046

<210> 3

<211> 682

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Met Gly Ser Ser His His His His His His His His His His Ala Val

1 5 10 15

Gly Gly Ser Gly Gly Ser Gly Gly Ser Tyr Lys Leu Ile Leu Asn Gly

20 25 30

Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr

35 40 45

Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly

50 55 60

Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Gly

65 70 75 80

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

85 90 95

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys His Met Pro

100 105 110

Arg Lys Arg Tyr Ser Cys Asp Phe Glu Thr Thr Thr Lys Val Glu Asp

115 120 125

Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile Glu Asp His Ser Glu

130 135 140

Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met Ala Trp Ala Leu Lys

145 150 155 160

Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys Phe Asp Gly Ala Phe

165 170 175

Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp Ser Ala Asp Gly

180 185 190

Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Thr Gly Gln Trp Tyr

195 200 205

Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg Lys Ile His Thr

210 215 220

Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe Pro Val Lys Lys Ile

225 230 235 240

Ala Lys Asp Cys Lys Leu Thr Val Leu Lys Gly Asp Ile Asp Tyr His

245 250 255

Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro Glu Glu Tyr Ala Tyr

260 265 270

Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu Leu Ile Gln Phe

275 280 285

Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser Asp Ser Leu Lys Asp

290 295 300

Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys Val Phe Pro Thr

305 310 315 320

Leu Ser Leu Gly Leu Asp Lys Lys Val Arg Tyr Ala Tyr Arg Gly Gly

325 330 335

Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu Ile Gly Glu Gly

340 345 350

Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala Gln Met Tyr Ser Arg

355 360 365

Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu Gly Lys Tyr Val Trp

370 375 380

Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile Arg Cys Glu Phe Glu

385 390 395 400

Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys Arg Ser Arg Phe

405 410 415

Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly Glu Ile Ala Asp

420 425 430

Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys Glu His Tyr Asp

435 440 445

Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe Lys Ala Thr Thr

450 455 460

Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr Tyr Ile Lys Thr Thr

465 470 475 480

Ser Glu Gly Cys Ile Lys Gln Leu Ala Lys Leu Met Leu Asn Ser Leu

485 490 495

Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr Gly Lys Val Pro Tyr

500 505 510

Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly Glu Glu Glu Thr

515 520 525

Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe Ile Thr Ala Trp Ala

530 535 540

Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr Asp Arg Ile Ile

545 550 555 560

Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr Glu Ile Pro Asp

565 570 575

Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly Tyr Trp Ala His

580 585 590

Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg Pro Lys Thr Tyr Ile

595 600 605

Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Glu Leu Val Glu Gly Ser

610 615 620

Pro Asp Asp Tyr Thr Asp Ile Lys Leu Ser Val Lys Cys Ala Gly Met

625 630 635 640

Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn Phe Lys Val Gly

645 650 655

Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val Pro Gly Gly Val

660 665 670

Val Leu Val Asp Asp Thr Phe Thr Ile Lys

675 680

<210> 4

<211> 574

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr Thr

1 5 10 15

Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile Glu

20 25 30

Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met Ala

35 40 45

Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys Phe

50 55 60

Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp

65 70 75 80

Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met

85 90 95

Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg

100 105 110

Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe Pro

115 120 125

Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly Asp

130 135 140

Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro Glu

145 150 155 160

Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu

165 170 175

Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser Asp

180 185 190

Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys

195 200 205

Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala

210 215 220

Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu

225 230 235 240

Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala Gln

245 250 255

Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu Gly

260 265 270

Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile Arg

275 280 285

Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys

290 295 300

Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly

305 310 315 320

Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys

325 330 335

Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe

340 345 350

Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr Tyr

355 360 365

Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu Met

370 375 380

Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr Gly

385 390 395 400

Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly

405 410 415

Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe Ile

420 425 430

Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr

435 440 445

Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr

450 455 460

Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly

465 470 475 480

Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg Gln

485 490 495

Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys Leu

500 505 510

Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys

515 520 525

Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn

530 535 540

Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val

545 550 555 560

Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys

565 570

Claims

1. A protein characterized in that: the protein is obtained by mutating phi29 DNA polymerase, the amino acid sequence of the phi29 DNA polymerase is shown as SEQ ID No.4, and the mutation is that phenylalanine at 136 th position of the phi29 DNA polymerase is mutated into cysteine and alanine at 376 th position of the phi29 DNA polymerase is mutated into cysteine.

2. A protein characterized in that: the amino acid sequence of the protein was compared with that of phi29 DNA polymerase, and the following mutations were performed: methionine at position 7 to arginine; valine at position 50 is mutated to alanine; methionine at position 96 is mutated to threonine; phenylalanine at position 136 is mutated to cysteine; glycine at position 196 is mutated to aspartic acid; the glutamic acid at position 220 is mutated to lysine; alanine at position 376 is mutated to cysteine; glutamine at position 496 is mutated to proline; lysine at position 511 is mutated to glutamic acid; phenylalanine at position 525 is mutated to leucine;

The amino acid sequence of phi29 DNA polymerase is shown as SEQ ID No. 4.

3. A fusion protein characterized in that: the amino acid sequence of the fusion protein is the amino acid sequence of the GB1 protein connected with the N end of the amino acid sequence of the protein in claim 1 or 2;

the amino acid sequence of the GB1 protein is shown in the 26 th-79 th positions of SEQ ID No. 3.

4. A fusion protein according to claim 3, wherein: the amino acid sequence of the fusion protein is shown as SEQ ID No. 3.

5. A biomaterial characterized in that: the biomaterial is any one of B1) to B4):

b1 A nucleic acid molecule encoding the protein of claim 1 or 2 or encoding the fusion protein of claim 3 or 4;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant microorganism comprising a nucleic acid molecule according to B1).

6. The biomaterial according to claim 5, wherein:

the nucleic acid molecule encoding the protein of claim 1 or 2 is any one of the following:

b11 The TTT at 406 th to 408 th positions of the nucleotide sequence of the optimized phi29 wild-type gene is replaced by TGC and the GCG at 1126 th to 1128 th positions is replaced by TGC, and other nucleotides are unchanged;

B12 The ATG at the 19 th to 21 th positions, the GTG at the 148 th to 150 th positions, the GCG at the 286 th to 288 th positions, the ACC at the 406 th to 408 th positions, the TGC at the 406 th to 588 th positions, the GAC at the 586 th to 588 th positions, the GAA at the 658 th to 660 th positions, the AAA at the 1126 th to 1128 th positions, the TGC at the 1126 th to 1488 th positions, the CCG at the 1486 th to 1488 th positions, the AAG at the 1531 th to 1533 th positions and the GAA are replaced, and other nucleotides are unchanged;

A dna replication method characterized by: comprising replicating DNA using the protein of claim 1 or 2 or the fusion protein of claim 3 or 4 as a DNA polymerase.

8. Use of a protein according to claim 1 or 2 or a fusion protein according to claim 3 or 4 as a DNA polymerase.

9. The use of a protein according to claim 1 or 2 or a fusion protein according to claim 3 or 4, either:

1) Performing DNA sequencing or RNA sequencing or whole genome sequencing;

2) RCA library establishment;

3) Genome amplification coverage detection;

4) Preparing a product for performing DNA sequencing or RNA sequencing or whole genome sequencing;

5) Preparing a product for RCA library establishment;

6) And preparing a product for genome amplification coverage detection.

10. Use of the biomaterial according to claim 5 or 6 for catalyzing DNA replication or for preparing a product for catalyzing DNA replication.

11. Use of the biomaterial according to claim 5 or 6, in any of the following:

1) Performing DNA sequencing or RNA sequencing or whole genome sequencing;

2) RCA library establishment;

3) Genome amplification coverage detection;

5) Preparing a product for RCA library establishment;

6) And preparing a product for genome amplification coverage detection.

12. A method for producing the protein of claim 1 or 2 or the fusion protein of claim 3 or 4, characterized in that: comprising the step of expressing a nucleic acid molecule encoding a protein according to claim 1 or 2 or a fusion protein according to claim 3 or 4 in an organism, said organism being a microorganism, a plant or a non-human animal, to obtain a protein according to claim 1 or 2 or a fusion protein according to claim 3 or 4.