CN112079903B - Mutant of mismatching binding protein and coding gene thereof - Google Patents

Abstract

The invention provides a mismatch binding protein eMuts, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID NO. 52 and has at least one mutation selected from the group consisting of: 1) Serine at positions 120 and 151 were mutated to cysteine; 2) Leucine at position 157 and glycine at position 233 were mutated to cysteine; 3) Glutamic acid at position 451 and valine at position 465 are mutated to cysteine; 4) The 609 th methionine and the 723 th threonine are mutated into cysteine; or 5) serine at position 120, leucine at position 157, glutamic acid at position 451, and methionine at position 609 are all mutated to cysteine. The mismatch binding protein provided by the invention has higher thermal stability, and can stably and specifically identify and bind to DNA containing an error sequence, thereby improving the accuracy of the DNA. The invention also provides host cells comprising a gene encoding the mismatch binding protein and methods of producing the mismatch binding protein using the same.

Description

A mutant of mismatch binding protein and its coding gene

technical field

The present invention relates to the field of biotechnology. Specifically, the present invention relates to a mutant of mismatch binding protein (abbreviated as eMutS) and its application.

Background technique

With the rise of synthetic biology, we have entered the era of creating genes with new gene functions and even whole genomes. DNA synthesis has a wide range of applications, including gene synthesis, metabolic pathway synthesis, and even whole genome synthesis (Gibson et al., 2008; Gibson et al., 2010). De novo gene synthesis, as an important technique in synthetic biology, mainly involves the assembly of short-chain oligonucleotides (oligouncletoides, oligos) into long-chain DNA by ligation reactions based on PCR or DNA ligase (Jingdong et al., 2004), Playing an increasingly important role in synthetic biology and biotechnology (Bayer and Smolke, 2005; Carr and Church, 2009; Gibson et al., 2008; Wang et al., 2009). Currently, oligonucleotides synthesized on porous silica beads are generally used as starting oligonucleotides for synthesis reactions, which are relatively expensive and have low synthesis throughput. Compared with traditional methods, the use of microfluidic synthesis technology to synthesize oligonucleotides in parallel on a solid phase plane (microfluidic chip) not only reduces the cost of oligonucleotides, but also greatly improves the throughput of synthesis (Gao et al. al., 2001; Quan et al., 2011; Richmond et al., 2004; Sriram et al., 2010; Xiaochuan et al., 2004). However, the efficiency of each step of the microchip synthesis reaction is lower than that of conventional methods, and has a relatively high error rate, resulting in microchip synthesis with lower synthesis quality and shorter oligonucleotide length (Tian et al. al., 2009). Errors in synthetic genes originate from erroneous bases introduced during chemical oligonucleotide synthesis, polymerase extension, and misassembly between oligonucleotides (Cline et al., 1996). Due to the existence of synthesis errors, it is usually unavoidable to select a clone with the correct sequence from multiple clones using clone sequencing methods, and the cost of this part usually occupies a larger part of the total cost of gene synthesis. Therefore, improving the fidelity of synthetic DNA is an urgent requirement for large-scale application of de novo gene synthesis technology. Enzymatic error correction methods are a cost-effective way to improve errors. Typically, these enzymes specifically recognize and bind DNA that contains mismatched sequences. Therefore, the DNA duplex is randomly combined with the wrong single strand and the correct single strand through denaturing annealing, so that the wrongly synthesized bases form a mismatch, and then use a mismatch binding protein (MutS) or a mismatch excision enzyme (for example, resolvase) Removed synthesis errors. Lubock et al. systematically compared six different error-correcting enzymes and concluded that MutS was the most suitable for increasing the number of perfect assemblies (up to 25.2-fold) (Lubock et al., 2017).

The Mismatch Repair (MMR) system ubiquitous in eukaryotes and prokaryotes ensures precise and stable replication of genetic material. The mismatch binding protein MutS is an important component of MMR, which recognizes and binds many different DNA mismatches (And and Jinksrobertson, 2000; Iyer et al., 2006; Modrich, 1991). Although the ability of the protein to bind different mismatches varies, it can bind almost all single-base mismatches as well as 1-4 base insertion or deletion mutations (Whitehouse et al., 1997). MutS derived from Escherichia coli (EcoMutS) is about 97 kDa and binds G:T, A:C, A:A, G:G more efficiently than other mismatches (Brown et al., 2001). MutS asymmetrically binds mismatched DNA as a homodimer.

MutS has been reported to be used to correct oligonucleotide-assembled gene fragments (Binkowski et al., 2005; Carret al., 2004). MutS from Thermus aquaticus (TaqMutS) is commonly used for error correction because it is more thermostable than EcoMutS and has weak binding capacity for fully complementary DNA. However, the binding ability of TaqMutS to mismatched bases is much weaker than that of EcoMutS, which directly reduces the efficiency of error correction (Brown et al., 2001; Cho et al., 2007). Recently, high-throughput error correction methods for correcting synthetic errors in ChIP-synthesized oligonucleotide libraries and their assembled genes were established (Wan et al., 2017; Wen et al., 2014). Using this method, the proteins EcoMutS and TaqMutS with mismatch binding ability can be immobilized on the cellulose column, which is low cost and high throughput. However, the poor thermal stability of EcoMutS limits the large-scale application of this error correction system in gene synthesis.

Therefore, in summary, there is an urgent need in the art for mismatch binding protein mutants with higher thermal stability.

Contents of the invention

The purpose of the present invention is to provide a mutant of mismatch binding protein and the application of the mutant.

In a first aspect, the invention provides a mismatch binding protein that:

a. comprising an amino acid sequence as shown in SEQ ID NO:52 and having a mutation selected from at least one of the following groups:

1) Both serine at position 120 and position 151 are mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 are both mutated to cysteine,

3) Both glutamic acid at position 451 and valine at position 465 are mutated into cysteine,

4) both methionine at position 609 and threonine at position 723 are mutated to cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451 and methionine at position 609 are all mutated to cysteine; or

b. The sequence defined in a) is formed by substitution, deletion or addition of 1-20 amino acid residues, and the mismatch binding protein derived from a) basically has the function of a mismatch binding protein as defined in a) protein. In a preferred embodiment, the mismatch binding protein is derived from bacteria of the genus Escherichia, preferably from Escherichia coli.

In a preferred embodiment, the mismatch binding protein:

a. have an amino acid sequence as shown in SEQ ID NO:52 and have a mutation selected from at least one group of the following:

1) Both serine at position 120 and position 151 are mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 are both mutated to cysteine,

3) Both glutamic acid at position 451 and valine at position 465 are mutated into cysteine,

4) both the methionine at position 609 and the threonine at position 723 mutations are cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451 and methionine at position 609 are all mutated to cysteine; or

b. have the sequence defined in a) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residues A mismatch binding protein derived from a) that is a sequence formed by substitution, deletion or addition of residues, and basically has the function of the mismatch binding protein defined in a).

In a preferred embodiment, the mismatch binding protein:

a. its amino acid sequence is shown in SEQ ID NO: 54, 56, 58, 60 or 62; or

b. comprising the sequence defined in a) through one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residues A mismatch binding protein derived from a) that is a sequence formed by substitution, deletion or addition of residues, and basically has the function of the mismatch binding protein defined in a).

In a preferred embodiment, the amino acid sequence of the mismatch binding protein is shown in SEQ ID NO: 54, 56, 58, 60 or 62.

In a preferred example, the mismatch binding protein retains at least 50% of its activity when stored at 4°C for 90 days; preferably, 60%-70% of its activity; more preferably, 70%-80% % or more activity, most preferably, more than 90% activity.

In a preferred example, the mismatch binding protein is purified using an immobilized column (MICC mismatch correction system) (Wen et al., 2014), which reduces the error rate of the synthetic gene to 2/kb, preferably, Reduced to 1/kb, most preferably, to 0.56/kb.

The term "corresponding to" used herein has a meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to the specified position in the other sequence.

In a second aspect, the present invention provides a gene encoding the protein described in the first aspect of the present invention.

In a preferred example, the nucleotide sequence of the gene is shown in SEQ ID NO: 53, 55, 57, 59 or 61.

In a third aspect, the present invention provides a vector comprising the coding gene of the second aspect of the present invention.

In the fourth aspect, the present invention provides a host cell containing the coding gene of the second aspect of the present invention.

In a preferred embodiment, the host cell is from the genus Escherichia, Corynebacterium, Brevibacterium sp., Bacillus, Serratia ( Serratia) or Vibrio.

In a preferred embodiment, the host cell is Escherichia coli (E. Coli).

In a preferred example, the host cell is chromosomally integrated with the coding gene of the second aspect of the present invention or contains the vector of the third aspect of the present invention.

In a preferred embodiment, said host cell expresses said mismatch binding protein.

In a fifth aspect, the present invention provides a method of preparing a mismatch binding protein, said method comprising the steps of:

a. cultivating the host cell according to the fourth aspect of the present invention to produce the mismatch binding protein; and

b. Isolating the mismatch binding protein from the culture medium.

In a preferred embodiment, the cultivation temperature of the method is 16-45°C, more preferably, the cultivation temperature is implemented at 16°C.

In the sixth aspect, the present invention provides the use of the mismatch binding protein described in the first aspect of the present invention in recognizing and binding to mismatched DNA.

In a preferred example, the mismatch binding protein described in the first aspect of the present invention is used in a free liquid state to recognize and bind to mismatched DNA or to combine with EGFP (Enhance green fluorescent protein, green fluorescent protein), CBM (Cellulosebinding module, cellulose structure) domain) to immobilize the mismatch binding protein on the column for recognition and binding to mismatched DNA. Preferably, the mismatch binding protein described in the first aspect of the present invention is used in combination with EGFP and CBM so that the immobilized mismatch binding protein has higher thermal stability on the column.

In the seventh aspect, the present invention provides a method for preparing the mismatch binding protein according to the first aspect of the present invention, the method comprising the following steps:

a. Transform the coding sequence of the amino acid sequence shown in SEQ ID NO:52, so that the amino acid sequence corresponding to SEQ ID NO:52 in the encoded amino acid sequence has at least one group of mutations selected from the following:

1) Both serine at position 120 and position 151 are mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 are both mutated to cysteine,

3) Both glutamic acid at position 451 and valine at position 465 are mutated into cysteine,

4) both the methionine at position 609 and the threonine at position 723 mutations are cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451 and methionine at position 609 are all mutated to cysteine;

b. directly transfecting the coding sequence obtained in a) into a suitable host cell or introducing a vector into a suitable host cell;

c. cultivating the host cell obtained in b);

d. isolating the mismatch binding protein produced by the host cell from the culture system obtained in step c); and

e. Determining the enzyme activity and thermal stability of the mismatch binding protein.

In an eighth aspect, the present invention provides a method for modifying a wild-type mismatch binding protein to improve thermal stability, the method comprising the following steps:

a. aligning the amino acid sequence of the wild-type mismatch binding protein with the amino acid sequence shown in SEQ ID NO:52; and

b. transforming the coding sequence of the wild-type mismatch binding protein, so that the amino acid sequence corresponding to SEQ ID NO:52 has a mutation selected from at least one of the following groups in the encoded amino acid sequence:

1) Both serine at position 120 and position 151 are mutated to cysteine,

2) Leucine at position 157 and glycine at position 233 are both mutated to cysteine,

3) Both glutamic acid at position 451 and valine at position 465 are mutated into cysteine,

4) both the methionine at position 609 and the threonine at position 723 mutations are cysteine, or

5) Serine at position 120, leucine at position 157, glutamic acid at position 451 and methionine at position 609 are all mutated to cysteine;

c. Directly transfecting the coding sequence obtained in b into a suitable host cell or introducing a vector into a suitable host cell;

d. culturing the host cells obtained in c;

e. isolating the mismatch binding protein produced by the host cell from the culture system obtained in step d; and

f. Determining the enzyme activity and thermal stability of the mismatch binding protein mutant.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the construction diagram of the plasmid in the present invention.

Figure 2 compares the thermostability at 4°C between the eMutS mutant of the present invention and the wild-type eMutS.

A. Results processed with the software "ImageJ".

According to the DNA brightness results on the DNA agarose gel, use the "ImageJ" software to give a RawIntDen value for all samples, and the RawIntDen ratio refers to the RawIntDen value of the sample/the RawIntDen value of the control eMutS, if the RawIntDen ratio is lower than 0.3, it means The protein is active; if the RawIntDen ratio is higher than 0.3, it means that the protein is inactive.

B. Thermostability of different eMutS protein mutants.

eMutS is the wild-type control, eMutS-E38C-G70C, eMutS-S120C-S151C, eMutS-L157C-G233C, eMutS-E177C-G195C, eMutS-Y474C-R500C, eMutS-E451C-V465C, eMutS-T581C-MutS-K644C, H585C-Q626C, eMutS-I597C-H760C, eMutS-M609C-T723C and eMutS-S120C-L157C-E451C-M609C are all different eMutS protein mutant samples.

Detailed ways

After extensive and in-depth research, the inventor unexpectedly found that the mismatch binding protein derived from Escherichia coli was subjected to site-directed mutation to increase the disulfide bond, and the obtained mismatch binding protein mutant could still maintain the protein after being stored at 4°C for 90 days. Greater than 90% activity. The present invention has been accomplished on this basis.

Application and advantage of the present invention:

1. Various mismatch binding proteins provided by the invention efficiently recognize and bind mismatched DNA;

2. The various mismatch binding proteins provided by the present invention have higher thermal stability than unmodified wild-type mismatch binding proteins, and have broad prospects for industrial application.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Reagents and bacterial strains: All reagents in the present invention are commercially purchased reagent grade reagents. Among them, Tryptone, Yeast extract, NaCl, plasmid extraction kit, gel recovery kit and all restriction endonucleases are from Shanghai Sangon Bioengineering Company. PrimeSTAR HS DNA polymerase, Solution I ligase and pMD18-T vector were purchased from Dalian Bao Biological Company. Escherichia coli XL10-gold strain was used as the host bacteria (Stratagene Company, California, USA) used for DNA manipulation, and Luria-Bertani (LB) medium containing 100 μg/ml ampicillin was used for culturing E. coli. The plasmid pEcoMutS-CBM3-EGFP (Wen et al., 2014) containing the wild-type eMutS gene sequence shown in SEQ ID NO: 51 was donated by a professor at the University of Science and Technology of China, and is currently kept in the laboratory of the inventor. LB medium (5g/l yeast extract, 10g/l tryptone, 5g/l Nacl) was used to induce expression medium.

The preparation of embodiment 1 bacterial strain:

1. Construct various plasmid vectors among the present invention:

1). Extract the plasmid of pEcoMutS-CBM3-EGFP (a gift from the professor of University of Science and Technology of China), and use the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primer E38C-G70C-F1 (SEQ ID No.01) and E38C-G70C-R1 (SEQ ID No.02) primers were used for PCR, and the amplified product was digested with DpnI to obtain a plasmid pZJQIBEBT001a with a site mutation. Then using the plasmid pZJQIBEBT001a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers E38C-G70C-F2 (SEQ ID No.03) and E38C-G70C-R2 (SEQ ID No.04) to carry out PCR amplification The pEcoMutS-E38C-G70C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT001.

The specific operation steps are:

First, extract the plasmid, the extraction steps are:

a. Add 2ml of LB medium containing corresponding antibiotics into a test tube with a capacity of 15ml, inoculate a single colony, seal it with a cotton plug, and culture overnight at 37°C with vigorous shaking (250rpm).

b. Transfer 1.5ml of the culture into a centrifuge tube and centrifuge at 4°C for 30 seconds (12,000×g) in a tabletop centrifuge.

c. Discard the supernatant, blot the liquid medium in the centrifuge tube with filter paper, suspend the bacterial sediment in 200 ml STET buffer, and mix well with a vortex mixer.

d. Add 4ml of freshly prepared lysozyme solution, mix well, and place at room temperature for 5 minutes.

e. Hold the centrifuge tube with a float, place it in a boiling water bath, accurately time it for 45 seconds, and centrifuge it for 10 minutes immediately after taking it out.

f. Use a sterile toothpick to pick up the sediment in the centrifuge tube and discard it. Add 8ml 5% CTAB to the supernatant of the centrifuge tube. After mixing with a mixer, centrifuge at high speed for 5min. Discard the supernatant and absorb it with filter paper. Dry the liquid in the centrifuge tube.

g. Add 300ml of 1.2M sodium chloride to fully dissolve the precipitate, then add 750ml of cold ethanol, mix thoroughly, centrifuge for 15min, and discard the supernatant.

h. Take 1ml of 70% cold ethanol, slowly rinse the inner wall of the centrifuge tube, and centrifuge for 5min. Discard the supernatant, blot the liquid on the tube wall with filter paper, and let the nucleic acid precipitate dry naturally at room temperature for 5-10 minutes.

i. Dissolve the precipitate in 50ml TE buffer solution and mix with a mixer. The plasmid preparation solution is ready, and the prepared plasmid is used for the next experiment.

The specific steps for obtaining mutant plasmids are as follows:

(1) PCR system for amplifying pEcoMutS-E38C-CBM3-EGFP full plasmid using pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-E38C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT001a.

①Enzyme digestion system of pEcoMutS-E38C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT001a (Fig. 1).

(3) A PCR system for amplifying the pEcoMutS-E38C-G70C-CBM3-EGFP full plasmid using the pZJQIBEBT001a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-E38C-G70C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT001.

① Enzyme digestion system of pEcoMutS-E38C-G70C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation at the second site was named pZJQIBEBT001 ( FIG. 1 ).

2). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers S120C-S151C-F1 (SEQ ID No.05) and S120C-S151C-R1 (SEQ ID No. 06) PCR was performed, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT002a. Then using the plasmid pZJQIBEBT002a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers S120C-S151C-F2 (SEQ ID No.07) and S120C-S151C-R2 (SEQ ID No.08) to carry out PCR amplification The pEcoMutS-S120C-S151C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT002.

The specific steps for obtaining mutant plasmids are as follows:

(1) PCR system for amplifying pEcoMutS-S120C-CBM3-EGFP full plasmid using pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-S120C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT002a.

①Enzyme digestion system of pEcoMutS-S120C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT002a ( FIG. 1 ).

(3) A PCR system for amplifying the pEcoMutS-S120C-S151C-CBM3-EGFP full plasmid using the pZJQIBEBT002a plasmid as a template:

PCR program

(4) Digest the entire plasmid of the amplified product pEcoMutS-S120C-S151C-CBM3-EGFP with Dpn I, and recover the product to obtain the second point-mutated plasmid pZJQIBEBT002.

① Enzyme digestion system of pEcoMutS-S120C-S151C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to the second site-directed mutation was named pZJQIBEBT002 ( FIG. 1 ).

3). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers L157C-G233C-F1 (SEQ ID No.09) and L157C-G233C-R1 (SEQ ID No. 10) PCR was performed, and the amplified product was digested with Dpn I to obtain a site-mutated plasmid pZJQIBEBT003a. Then using the plasmid pZJQIBEBT003a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers L157C-G233C-F2 (SEQ ID No.11) and L157C-G233C-R2 (SEQ ID No.12) to carry out PCR amplification The pEcoMutS-L157C-G233C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT003.

The specific steps for obtaining mutant plasmids are as follows:

(1) The PCR system for amplifying the pEcoMutS-L157C-CBM3-EGFP full plasmid using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-L157C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT003a.

①Enzyme digestion system of pEcoMutS-L157C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT003a (Fig. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-L157C-G233C-CBM3-EGFP using the pZJQIBEBT003a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-L157C-G233C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT003.

① Enzyme digestion system of pEcoMutS-L157C-G233C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation at the second site was named pZJQIBEBT003 ( FIG. 1 ).

4). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers E177C-G195C-F1 (SEQ ID No.13) and E177C-G195C-R1 (SEQ ID No. 14) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT004a with a site mutation. Then using the plasmid pZJQIBEBT004a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers E177C-G195C-F2 (SEQ ID No.15) and E177C-G195C-R2 (SEQ ID No.16) to carry out PCR amplification The pEcoMutS-E177C-G195C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT004.

The specific steps for obtaining mutant plasmids are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-E177C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-E177C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT004a.

①Enzyme digestion system of pEcoMutS-E177C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT004a (Fig. 1).

(3) A PCR system for amplifying the entire plasmid pEcoMutS-E177C-G195C-CBM3-EGFP using the pZJQIBEBT004a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-E177C-G195C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT004.

① Enzyme digestion system of pEcoMutS-E177C-G195C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation at the second site was named pZJQIBEBT004 ( FIG. 1 ).

5). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers E451C-V465C-F1 (SEQ ID No.17) and E451C-V465C-R1 (SEQ ID No. 18) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT005a with a site mutation. Then using the plasmid pZJQIBEBT005a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers E451C-V465C-F2 (SEQ ID No.19) and E451C-V465C-R2 (SEQ ID No.20) to carry out PCR amplification The whole pEcoMutS-E451C-V465C-CBM3-EGFP plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT005.

The specific steps for obtaining mutant plasmids are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-E451C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-E451C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT005a.

①Enzyme digestion system of pEcoMutS-E451C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT005a (Fig. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-E451C-V465C-CBM3-EGFP using the pZJQIBEBT005a plasmid as a template:

PCR program

(4) Digest the entire plasmid of the amplified product pEcoMutS-E451C-V465C-CBM3-EGFP with Dpn I, and recover the product to obtain the second point-mutated plasmid pZJQIBEBT005.

① Enzyme digestion system of pEcoMutS-E451C-V465C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation at the second site was named pZJQIBEBT005 ( FIG. 1 ).

6). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers Y474C-R500C-F1 (SEQ ID No.21) and Y474C-R500C-R1 (SEQ ID No. 22) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT006a with a site mutation. Then using the plasmid pZJQIBEBT006a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers Y474C-R500C-F2 (SEQ ID No.23) and Y474C-R500C-R2 (SEQ ID No.24) to carry out PCR amplification The pEcoMutS-Y474C-R500C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT006.

The specific steps for obtaining mutant plasmids are as follows:

(1) PCR system for amplifying the whole plasmid pEcoMutS-Y474C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-Y474C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT006a.

①Enzyme digestion system of pEcoMutS-Y474C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT006a ( FIG. 1 ).

(3) PCR system for amplifying the whole plasmid pEcoMutS-Y474C-R500C-CBM3-EGFP using the pZJQIBEBT006a plasmid as a template:

PCR program

(4) Digest the entire plasmid of the amplified product pEcoMutS-Y474C-R500C-CBM3-EGFP with Dpn I, and recover the product to obtain the second point-mutated plasmid pZJQIBEBT006.

① Enzyme digestion system of pEcoMutS-Y474C-R500C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to the second site-directed mutation was named pZJQIBEBT006 ( FIG. 1 ).

7). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers T581C-K644C-F1 (SEQ ID No.25) and T581C-K644C-R1 (SEQ ID No. 26) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT007a with a site mutation. Then using the plasmid pZJQIBEBT007a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers T581C-K644C-F2 (SEQ ID No.27) and T581C-K644C-R2 (SEQ ID No.28) to carry out PCR amplification The whole pEcoMutS-T581C-K644C-CBM3-EGFP plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT007.

The specific steps for obtaining mutant plasmids are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-T581C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) Digest the entire plasmid of the amplified product pEcoMutS-T581C-CBM3-EGFP with Dpn I, and recover the product to obtain the first point-mutated plasmid pZJQIBEBT007a.

①Enzyme digestion system of pEcoMutS-T581C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT007a (Fig. 1).

(3) PCR system for amplifying the whole plasmid pEcoMutS-T581C-K644C-CBM3-EGFP using the pZJQIBEBT007a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-T581C-K644C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT007.

① Enzyme digestion system of pEcoMutS-T581C-K644C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation at the second site was named pZJQIBEBT007 ( FIG. 1 ).

8). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers H585C-Q626C-F1 (SEQ ID No.29) and H585C-Q626C-R1 (SEQ ID No. 30) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT008a with a site mutation. Then using the plasmid pZJQIBEBT008a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers H585C-Q626C-F2 (SEQ ID No.31) and H585C-Q626C-R2 (SEQ ID No.32) to carry out PCR amplification The pEcoMutS-H585C-Q626C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, which was named pZJQIBEBT008.

The specific steps for obtaining mutant plasmids are as follows:

(1) PCR system for amplifying the whole plasmid pEcoMutS-H585C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-H585C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT008a.

①Enzyme digestion system of pEcoMutS-H585C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT008a (Fig. 1).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-H585C-Q626C-CBM3-EGFP using the pZJQIBEBT008a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-H585C-Q626C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT008.

① Enzyme digestion system of pEcoMutS-H585C-Q626C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to the second site-directed mutation was named pZJQIBEBT008 ( FIG. 1 ).

9). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers I597C-H760C-F1 (SEQ ID No.33) and I597C-H760C-R1 (SEQ ID No. 34) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT009a with a site mutation. Then using the plasmid pZJQIBEBT009a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers I597C-H760C-F2 (SEQ ID No.35) and I597C-H760C-R2 (SEQ ID No.36) to carry out PCR amplification The pEcoMutS-I597C-H760C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, named pZJQIBEBT009.

The specific steps for obtaining mutant plasmids are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-I597C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-I597C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT009a.

①Enzyme digestion system of pEcoMutS-I597C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT009a ( FIG. 1 ).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-I597C-H760C-CBM3-EGFP using the pZJQIBEBT009a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-I597C-H760C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT009.

①Enzyme digestion system of pEcoMutS-I597C-H760C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to the second site-directed mutation was named pZJQIBEBT009 ( FIG. 1 ).

10). Taking the extracted pEcoMutS-CBM3-EGFP plasmid as a template, using KOD Plus Neo DNA polymerase (TOYOBO), primers M609C-T723C-F1 (SEQ ID No.37) and M609C-T723C-R1 (SEQ ID No. 38) PCR was performed, and the amplified product was digested with Dpn I to obtain a plasmid pZJQIBEBT010a with a site mutation. Then, using the plasmid pZJQIBEBT010a as a template, use KOD Plus Neo DNA polymerase (TOYOBO), primers M609C-T723C-F2 (SEQ ID No.39) and M609C-T723C-R2 (SEQ ID No.40) to carry out PCR amplification The pEcoMutS-M609C-T723C-CBM3-EGFP complete plasmid was digested with Dpn I to obtain a plasmid with two mutations, named pZJQIBEBT010.

The specific steps for obtaining mutant plasmids are as follows:

(1) The PCR system for amplifying the whole plasmid pEcoMutS-M609C-CBM3-EGFP using the pEcoMutS-CBM3-EGFP plasmid as a template:

PCR program

(2) The entire plasmid of the amplified product pEcoMutS-M609C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the first point-mutated plasmid pZJQIBEBT010a.

①Enzyme digestion system of pEcoMutS-M609C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to site-directed mutation was named pZJQIBEBT010a ( FIG. 1 ).

(3) The PCR system for amplifying the whole plasmid pEcoMutS-M609C-T723C-CBM3-EGFP using the pZJQIBEBT010a plasmid as a template:

PCR program

(4) The entire plasmid of the amplified product pEcoMutS-M609C-T723C-CBM3-EGFP was digested with Dpn I, and the product was recovered to obtain the second point-mutated plasmid pZJQIBEBT010.

① Enzyme digestion system of pEcoMutS-M609C-T723C-CBM3-EGFP whole plasmid:

② The obtained plasmid subjected to the second site-directed mutation was named pZJQIBEBT010 ( FIG. 1 ).

10). Taking the extracted pEcoMutS-CBM3-EGFP plasmid, pZJQIBEBT002 plasmid, pZJQIBEBT003 plasmid, pZJQIBEBT006 plasmid, and pZJQIBEBT010 plasmid as templates respectively, using KOD Plus Neo DNA polymerase (TOYOBO), primer eMutS-NheI-F (SEQ ID No. 41) and eMutS-340-R (SEQ ID No.42), eMutS-310-F (SEQ ID No.43) and eMutS-S120C-460-R (SEQ ID No.44), eMutS-430-F ( SEQ ID No.45) and eMutS-800-R (SEQ ID No.46), eMutS-770-F (SEQ ID No.47) and eMutS-1500-R (SEQ ID No.48), eMutS-1470-F (SEQ ID No.49) and eMutS-SacI-R (SEQ ID No.50) carry out PCR, obtain product eMutS-1-340, S120C-310-460, L157C-430-800, E451C-770-1500, M609C -1470- end. The amplified product is then utilized for fusion PCR with KOD Plus Neo DNA polymerase (TOYOBO), primers eMutS-NheI-F (SEQ ID No.41) and eMutS-SacI-R (SEQ ID No.50), to obtain the product eMutS- S120C-L157C-E451C-M609C. The fragment eMutS-S120C-L157C-E451C-M609C and the plasmid pEcoMutS-CBM3-EGFP were respectively digested with NheI and SacI, ligated, and a plasmid with eight mutations was obtained after transformation, which was named pZJQIBEBT011 (Figure 1).

The specific steps for obtaining mutant plasmids are as follows:

(1) Using the pEcoMutS-CBM3-EGFP plasmid as a template, eMutS-NheI-F (SEQ ID No.41) and eMutS-340-R (SEQ ID No.42) as primers, amplify the eMutS-1-340 fragment PCR system:

PCR program

(2) Using the pZJQIBEBT002 plasmid as a template, eMutS-310-F (SEQ ID No.43) and eMutS-S120C-460-R (SEQ ID No.44) as primers, a PCR system for amplifying the eMutS-1-340 fragment :

PCR program

(3) Using the pZJQIBEBT003 plasmid as a template, eMutS-430-F (SEQ ID No.45) and eMutS-800-R (SEQ ID No.46) as primers, a PCR system for amplifying the L157C-430-800 fragment:

PCR program

(4) Using the pZJQIBEBT006 plasmid as a template, eMutS-770-F (SEQ ID No.47) and eMutS-1500-R (SEQ ID No.48) as primers, a PCR system for amplifying the E451C-770-1500 fragment:

PCR program

(5) Using the pZJQIBEBT010 plasmid as a template, eMutS-1470-F (SEQ ID No.49) and eMutS-SacI-R (SEQ ID No.50) as primers, the PCR system for amplifying the M609C-1470-end fragment:

PCR program

(6) Then the amplified product was carried out fusion PCR using KOD Plus Neo DNA polymerase (TOYOBO), primers eMutS-NheI-F (SEQ ID No.41) and eMutS-SacI-R (SEQ ID No.50), to obtain The PCR system of the product eMutS-S120C-L157C-E451C-M609C:

PCR program

(7) The fragment eMutS-S120C-L157C-E451C-M609C and the plasmid pEcoMutS-CBM3-EGFP were respectively digested with NheI and SacI, ligated, and a plasmid with eight mutations was obtained after transformation, which was named pZJQIBEBT011 (Figure 1).

① Enzyme digestion system for fragment eMutS-S120C-L157C-E451C-M609C:

② Enzyme digestion system of plasmid pEcoMutS-CBM3-EGFP:

③ The digested products were ligated using Solution I (TAKATA), and the obtained plasmid containing 8 site-directed mutations was named pZJQIBEBT011 (Figure 1).

2. Transform the constructed vector into the expression vector Escherichia coli BL21:

The preparation of the host strain Escherichia coli BL21 by transforming the 10 plasmids that have undergone site-directed mutation

1) Escherichia coli chemical transformation steps:

①. Take out a tube (100ul) of competent bacteria from the ultra-low temperature freezer at -70°C, heat it with your fingers immediately to melt it, insert it on ice, and bathe in ice for 5-10 minutes. .

②. Add 5ul of the ligated plasmid mixture (DNA content does not exceed 100ng), shake gently and place on ice for 20min.

③. Gently shake well and insert into a 42°C water bath for 45 seconds for heat shock, then quickly put it back into the ice, and let it stand for 3-5 minutes.

④. Add 500ul LB medium (without antibiotics) to each of the above-mentioned tubes in the ultra-clean workbench and mix gently, then fix it on the spring stand of the shaker at 37°C and shake for 1h.

⑤. Take 100-300 ul of the above-mentioned transformation mixture in the ultra-clean workbench, drop them into solid LB plate culture dishes containing appropriate antibiotics, and spread evenly with a glass coating rod burned by an alcohol lamp.

⑥. Mark the coated culture dish, place it in a 37°C constant temperature incubator for 30-60 minutes until the liquid on the surface penetrates into the medium, then turn it upside down and put it in a 37°C constant temperature incubator overnight.

⑦. Pick the clones on the plate and culture them in liquid LB, extract the plasmids, and identify the transformation results by PCR.

2) The specific process of constructing various eMutS protein mutant expression strains:

The plasmid pZJQIBEBT001 containing mutations at E38C and G70C was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT001;

The plasmid pZJQIBEBT002 containing the S120C and S151C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT002;

The plasmid pZJQIBEBT003 containing the L157C and G233C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT003;

The plasmid pZJQIBEBT004 containing the E177C and G195C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT004;

The plasmid pZJQIBEBT005 containing mutations at E451C and V465C was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT005;

The plasmid pZJQIBEBT006 containing Y474C and R500C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT006;

The plasmid pZJQIBEBT007 containing T581C and K644C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT007;

The plasmid pZJQIBEBT008 containing the H585C and Q626C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT008;

The plasmid pZJQIBEBT009 containing the I597C and H760C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT009;

The plasmid pZJQIBEBT010 containing the M609C and T723C mutations was transformed into BL21 Escherichia coli. The strain will be restored to function with Amp resistance and added to the function of eMutS. Positive clones were screened on a medium containing Amp resistance (recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l), and the resulting transformed strain was named: EZJQIBEBT010;

3) extract the plasmid, and identify the positive strain transformed by Escherichia coli by PCR.

(1) Escherichia coli plasmid extraction steps:

①. Bacteria collection: Centrifuge the overnight culture (37°C, 12-16 hours) at room temperature≧10,000g for 1-2 minutes, and discard the supernatant completely.

②. Resuspension: Add 250 μl of RNase A-containing cell suspension (S1), fully suspend and shake or beat repeatedly with a pipette tip to completely disperse and suspend the bacteria.

③. Lysis: Add 250 μl of cell lysate (S2), mix gently up and down 5 times, and let stand at room temperature for 1-5 minutes. After the bacteria are fully lysed, the solution becomes translucent.

④. Neutralization: Add 350 μl neutralization buffer (S3), mix gently up and down 5 times, mix well, avoid violent shock. Centrifuge at ≧12,000g for 10 minutes at room temperature.

⑤. DNA binding: Carefully aspirate the supernatant, transfer it to a centrifugal adsorption column inserted into a collection tube, centrifuge at ≧12,000g for 1 minute at room temperature, discard the waste liquid in the collection tube, and reinsert the centrifugal adsorption column into the collection tube.

⑥. Cleaning: Add 500μl of washing solution (WB, please confirm that ethanol has been added!) to the centrifugal adsorption column, centrifuge at ≧12,000g for 30 seconds at room temperature, discard the waste liquid in the collection tube, and insert the centrifugal adsorption column back to collect tube.

⑦. Cleaning: Add 500 μl of washing solution (WB, please confirm that ethanol has been added!) to the centrifugal adsorption column, centrifuge at ≧12,000g for 30 seconds at room temperature, discard the waste liquid in the collection tube, and insert the centrifugal adsorption column back to collect tube. Uncover the centrifugal adsorption column and centrifuge again for 2 minutes to completely remove the residual rinse solution. .

⑧. Elution: Carefully take out the centrifugal adsorption column and insert it into a new 1.5ml sterilized centrifuge tube. Add 100 μl of elution buffer (EB) to the center of the silica gel adsorption membrane, and after standing at room temperature for 1 minute, centrifuge at ≧12,000 g for 1 minute to collect plasmid DNA.

⑨. Storage: Discard the centrifugal adsorption column, and the purified plasmid can be used directly for subsequent reactions or stored at -20°C for a long time.

(2) PCR system for identifying positive strains transformed by E. coli:

①Plasmid PCR system containing eMutS:

PCR program

The general primer M13 is used as a primer, and the plasmid is used as a template. After PCR amplification, the strain that can specifically amplify the gene band is a positive strain, and the next step of the experiment is carried out. The size of the gene is 2586bp.

The thermostability of embodiment 2eMutS protein mutant

This example is used to compare the stability of protease activity expressed by strains of different eMutS mutants. The results showed that the protein eMutS-S120C-L157C-E451C-M609C expressed by EZJQIBEBT011 strain had the highest activity and stability.

1. Recover strains on LB medium plates. Control strain: eMutS. Experimental strains: eMutS-E38C-G70C, eMutS-Y474C-R500C, eMutS-T581C-K644C, eMutS-I597C-H760C, eMutS-S120C-S151C, eMutS-L157C-G233C, eMutS-E451C-V465C, eMutS-T79CutS-M70 , eMutS-E177C-G195C, eMutS-H585C-Q626C, eMutS-S120C-L157C-E451C-M609C. Incubate at 37°C for 1 day.

2. Pick out single clones respectively, and connect them to 5ml liquid LB medium. 37°C, 250rpm, overnight.

3. Prepare 36 bottles of 100mL YPD medium and distribute them in 1L Erlenmeyer flasks. Recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l. Sterilized and ready to use.

4. Take an appropriate amount of overnight culture and inoculate it into 200ml LB medium at an inoculation ratio of 1:1000, culture at 37°C and 250rpm.

5. When OD600=0.8 or so, harvest the bacteria, crush the bacteria, purify the protein, and measure the enzyme activity.

6. It can be seen from Figure 2 that the mutants eMutS-E38C-G70C and eMutS-Y474C-R500C lost enzyme activity. The enzyme activity of mutants eMutS-T581C-K644C and eMutS-I597C-H760C can last for 21 days, which is similar to that of the control group. Mutants eMutS-S120C-S151C, eMutS-L157C-G233C, eMutS-E451C-V465C and eMutS-M609C-T723C, eMutS-S120C-L157C-E451C-M609C were longer than the control group, and the mutant eMutS -S120C-L157C-E451C-M609C can last for up to 90 days.

Example 3 Error Correction Ability of eMutS Protein Mutants in Binding to Mismatched DNA

This example is used to verify the error correction ability of the protease expressed by the strains of the two eMutS mutants with the strongest enzyme activity stability. The results showed that the error-correcting ability of proteases with improved stability was still maintained. Among them, eMutS-S120C-L157C-E451C-M609C has the best error correction effect, even better than the strain before mutation.

1. Recover strains on LB medium plates. Control strain: eMutS. Experimental strains: eMutS-S120C-S151C, eMutS-L157C-G233C, eMutS-S120C-L157C-E451C-M609C. Incubate at 37°C for 1 day.

2. Pick out single clones respectively, and connect them to 5ml liquid LB medium. 37°C, 250rpm, overnight.

3. Prepare 12 bottles of 100mL YPD medium and distribute them in 1L Erlenmeyer flasks. Recipe: tryptone 10g/l, yeast extract 5g/l, NaCl 10g/l. Sterilized and ready to use.

4. Take an appropriate amount of overnight culture and inoculate it into 200ml LB medium at an inoculation ratio of 1:1000, culture at 37°C and 250rpm.

5. When OD600=0.8 or so, harvest the bacteria, crush the bacteria, purify the protein, and use it for the DNA error correction experiment.

6. In applied experiments, all eMutS proteins were used with the MICC system, which were reported in past studies. Error correction of XR and Cas9 homologs synthesized with MCp-oligos using the error correction system with improved stability. As shown in Table 1, the oligomer error rate is about 1.2%. Removing errors using MICC significantly reduced the error rate for synthesizing XR and Cas9 homologs. Experiments show that the construction of disulfide bonds improves the stability of eMutS protein while maintaining the original error correction ability. Among them, eMutS-S120C-L157C-E451C-M609C not only greatly improved thermal stability, but also had the best error correction effect, even better than the strain before mutation.

Table 1. Results of DNA error correction in eMutS mutants

It should be understood that while the invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that, without departing from the spirit and scope of the invention as defined by the appended claims, , various changes in form and details can be made therein, and any combination of various embodiments can be made.

sequence listing

<110> Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences

<120> A mutant of a mismatch binding protein and its coding gene

<130> P2019-0992

<160> 62

<170> SIPOSequenceListing 1.0

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ggatgggtga tttttattgc ctgttttatg acgacg 36

<210> 2

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<212>DNA

<213> Artificial sequence (Artificial sequence)

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cgtcgtcata aaacaggcaa taaaaatcac ccatcc 36

<210> 3

<211> 35

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gccgatcccg atggcgtgca ttccctacca tgcgg 35

<210> 4

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<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ccgcatggta gggaatgcac gccatcggga tcggc 35

<210> 5

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<212>DNA

<213> Artificial sequence (Artificial sequence)

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tacgccaggc accatctgcg atgaagccct gttgc 35

<210> 6

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<213> Artificial sequence (Artificial sequence)

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gcaacagggc ttcatcgcag atggtgcctg gcgta 35

<210> 7

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<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

gcgacgctgg atatctgctc cgggcgtttt cgc 33

<210> 8

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<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

gcgaaaacgc ccggagcaga tatccagcgt cgc 33

<210> 9

<211> 35

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

ttccgggcgt tttcgctgca gcgaaccggc tgacc 35

<210> 10

<211> 35

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

ggtcagccgg ttcgctgcag cgaaaacgcc cggaa 35

<210> 11

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<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

cgagaacgcg ccgcgctgcc tttgtgctgc cggttg 36

<210> 12

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<213> Artificial sequence (Artificial sequence)

<400> 12

caaccggcag cacaaaggca gcgcggcgcg ttctcg 36

<210> 13

<211> 35

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

acgcactaat cctgcgtgcc tgctgtatgc agaag 35

<210> 14

<211> 35

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

cttctgcata cagcaggcac gcaggattag tgcgt 35

<210> 15

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<213> Artificial sequence (Artificial sequence)

<400> 15

attgaaggcc gtcgctgcct gcgccgtcgc ccg 33

<210> 16

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<213> Artificial sequence (Artificial sequence)

<400> 16

cgggcgacgg cgcaggcagc gacggccttc aat 33

<210> 17

<211> 34

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

tatctggagc gtctgtgcgt ccgcgagcgt gaac 34

<210> 18

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<213> Artificial sequence (Artificial sequence)

<400> 18

gttcacgctc gcggacgcac agacgctcca gata 34

<210> 19

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<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

ctggacacgc tgaaatgcgg ctttaatgcg gtg 33

<210> 20

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

caccgcatta aagccgcatt tcagcgtgtc cag 33

<210> 21

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gcggtgcacg gctactgcat tcaaatcagc cgt 33

<210> 22

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

acggctgatt tgaatgcagt agccgtgcac cgc 33

<210> 23

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

ctgaaaaacg ccgagtgcta catcattcca gag 33

<210> 24

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

ctctggaatg atgtagcact cggcgttttt cag 33

<210> 25

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

ccgggcattc gcatttgcga aggtcgccat ccg 33

<210> 26

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

cggatggcga ccttcgcaaa tgcgaatgcc cgg 33

<210> 27

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

tatgtaccgg cacaatgcgt cgagattgga cct 33

<210> 28

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

aggtccaatc tcgacgcatt gtgccggtac ata 33

<210> 29

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

attaccgaag gtcgctgccc ggtagttgaa caa 33

<210> 30

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

ttgttcaact accgggcagc gaccttcggt aat 33

<210> 31

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

agtacctata tgcgctgcac cgcactgatt gcg 33

<210> 32

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

cgcaatcagt gcggtgcagc gcatataggt act 33

<210> 33

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

ctgaatgagc cattttgcgc caacccgctg aat 33

<210> 34

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

attcagcggg ttggcgcaaa atggctcatt cag 33

<210> 35

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

accattgcct ttatgtgcag cgtgcaggat ggc 33

<210> 36

<211> 33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

gccatcctgc acgctgcaca taaaggcaat ggt 33

<210> 37

<211> 34

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

tcgccgcagc gccgctgctt gatcatcacc ggtc 34

<210> 38

<211> 34

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

gaccggtgat gatcaagcag cggcgctgcg gcga 34

<210> 39

<211> 36

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

taagattaag gcattgtgct tattgctac cacta 36

<210> 40

<211> 36

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

tagtgggtag caaataagca caatgcctta atctta 36

<210> 41

<211> 31

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

ctagctagca tgagtgcaat agaaaatttc g 31

<210> 42

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

cgatacgcac aactttgcgc 20

<210> 43

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

gtccggttga gcgcaaagtt 20

<210> 44

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

gcccggagca gatatccagc 20

<210> 45

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

gctacgcgac gctggatatc 20

<210> 46

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

atgctgtcct gctcacgttc 20

<210> 47

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

catcaccatg gaacgtgagc 20

<210> 48

<211> 20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

gcgctcggcg tttttcagcg 20

<210> 49

<211> 22

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

cgtcgccaga cgctgaaaaa cg 22

<210> 50

<211> 25

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

cgagctccac caggctcttc aagcg 25

<210> 51

<211> 2562

<212>DNA

<213> Escherichia coli

<400> 51

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcggggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatcagc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc agttccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact aattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccattat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggatta tcgcttgaag agcctggtgt aa 2562

<210> 52

<211> 853

<212> PRT

<213> Escherichia coli

<400> 52

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

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

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 53

<211> 2562

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcggggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact aattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccattat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggatta tcgcttgaag agcctggtgt aa 2562

<210> 54

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 54

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

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

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Cys Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Cys Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 55

<211> 2562

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcggggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgctg cagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgctgcc tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact aattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccattat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggatta tcgcttgaag agcctggtgt aa 2562

<210> 56

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 56

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

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

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Cys Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Cys Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 57

<211> 2562

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcggggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg tgcgtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aatgcggctt taatgcggtg cacggctact aattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccattat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgcatgttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgacgt tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggatta tcgcttgaag agcctggtgt aa 2562

<210> 58

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 58

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

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

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Cys Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Cys Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Met Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Thr Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 59

<211> 2562

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcggggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgcct gagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgcggac tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg gaagtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aagttggctt taatgcggtg cacggctact aattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccattat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgctgcttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgtgct tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggatta tcgcttgaag agcctggtgt aa 2562

<210> 60

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 60

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

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

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Ser Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Ser Ser Gly Arg Phe Arg Leu Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Gly Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Glu Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Val Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Cys Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Cys Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

<210> 61

<211> 2562

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

atgagtgcaa tagaaaattt cgacgcccat acgcccatga tgcagcagta tctcaggctg 60

aaagcccagc atcccgagat cctgctgttt taccggatgg gtgattttta tgaactgttt 120

tatgacgacg caaaacgcgc gtcgcaactg ctggatattt cactgaccaa acgcggtgct 180

tcggcggggag agccgatccc gatggcgggg attccctacc atgcggtgga aaactatctc 240

gccaaactgg tgaatcaggg agagtccgtt gccatctgcg aacaaattgg cgatccggcg 300

accagcaaag gtccggttga gcgcaaagtt gtgcgtatcg ttacgccagg caccatctgc 360

gatgaagccc tgttgcagga gcgtcaggac aacctgctgg cggctatctg gcaggacagc 420

aaaggtttcg gctacgcgac gctggatatc tgctccgggc gttttcgctg cagcgaaccg 480

gctgaccgcg aaacgatggc ggcagaactg caacgcacta atcctgcgga actgctgtat 540

gcagaagatt ttgctgaaat gtcgttaatt gaaggccgtc gcggcctgcg ccgtcgcccg 600

ctgtgggagt ttgaaatcga caccgcgcgc cagcagttga atctgcaatt tgggacccgc 660

gatctggtcg gttttggcgt cgagaacgcg ccgcgctgcc tttgtgctgc cggttgtctg 720

ttgcagtatg cgaaagatac ccaacgtacg actctgccgc atattcgttc catcaccatg 780

gaacgtgagc aggacagcat cattatggat gccgcgacgc gtcgtaatct ggaaatcacc 840

cagaacctgg cgggtggtgc ggaaaatacg ctggcttctg tgctcgactg caccgtcacg 900

ccgatgggca gccgtatgct gaaacgctgg ctgcatatgc cagtgcgcga tacccgcgtg 960

ttgcttgagc gccagcaaac tattggcgca ttgcaggatt tcaccgccgg gctacagccg 1020

gtactgcgtc aggtcggcga cctggaacgt attctggcac gtctggcttt acgaactgct 1080

cgcccacgcg atctggcccg tatgcgccac gctttccagc aactgccgga gctgcgtgcg 1140

cagttagaaa ctgtcgatag tgcaccggta caggcgctac gtgagaagat gggcgagttt 1200

gccgagctgc gcgatctgct ggagcgagca atcatcgaca caccgccggt gctggtacgc 1260

gacggtggtg ttatcgcatc gggctataac gaagagctgg atgagtggcg cgcgctggct 1320

gacggcgcga ccgattatct ggagcgtctg tgcgtccgcg agcgtgaacg taccggcctg 1380

gacacgctga aatgcggctt taatgcggtg cacggctact aattcaaat cagccgtggg 1440

caaagccatc tggcacccat caactacatg cgtcgccaga cgctgaaaaa cgccgagcgc 1500

tacatcattc cagagctaaa agagtacgaa gataaagttc tcacctcaaa aggcaaagca 1560

ctggcactgg aaaaacagct ttatgaagag ctgttcgacc tgctgttgcc gcatctggaa 1620

gcgttgcaac agagcgcgag cgcgctggcg gaactcgacg tgctggttaa cctggcggaa 1680

cgggcctata ccctgaacta cacctgcccg accttcattg ataaaccggg cattcgcatt 1740

accgaaggtc gccatccggt agttgaacaa gtactgaatg agccattat cgccaacccg 1800

ctgaatctgt cgccgcagcg ccgctgcttg atcatcaccg gtccgaacat gggcggtaaa 1860

agtacctata tgcgccagac cgcactgatt gcgctgatgg cctacatcgg cagctatgta 1920

ccggcacaaa aagtcgagat tggacctatc gatcgcatct ttacccgcgt aggcgcggca 1980

gatgacctgg cgtccgggcg ctcaaccttt atggtggaga tgactgaaac cgccaatatt 2040

ttacataacg ccaccgaata cagtctggtg ttaatggatg agatcgggcg tggaacgtcc 2100

acctacgatg gtctgtcgct ggcgtgggcg tgcgcggaaa atctggcgaa taagattaag 2160

gcattgtgct tatttgctac ccactatttc gagctgaccc agttaccgga gaaaatggaa 2220

ggcgtcgcta acgtgcatct cgatgcactg gagcacggcg acaccattgc ctttatgcac 2280

agcgtgcagg atggcgcggc gagcaaaagc tacggcctgg cggttgcagc tctggcaggc 2340

gtgccaaaag aggttattaa gcgcgcacgg caaaagctgc gtgagctgga aagcatttcg 2400

ccgaacgccg ccgctacgca agtggatggt acgcaaatgt ctttgctgtc agtaccagaa 2460

gaaacttcgc ctgcggtcga agctctggaa aatcttgatc cggattcact caccccgcgt 2520

caggcgctgg agtggatta tcgcttgaag agcctggtgt aa 2562

<210> 62

<211> 853

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 62

Met Ser Ala Ile Glu Asn Phe Asp Ala His Thr Pro Met Met Gln Gln

1 5 10 15

Tyr Leu Arg Leu Lys Ala Gln His Pro Glu Ile Leu Leu Phe Tyr Arg

20 25 30

Met Gly Asp Phe Tyr Glu Leu Phe Tyr Asp Asp Ala Lys Arg Ala Ser

35 40 45

Gln Leu Leu Asp Ile Ser Leu Thr Lys Arg Gly Ala Ser Ala Gly Glu

50 55 60

Pro Ile Pro Met Ala Gly Ile Pro Tyr His Ala Val Glu Asn Tyr Leu

65 70 75 80

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

85 90 95

Gly Asp Pro Ala Thr Ser Lys Gly Pro Val Glu Arg Lys Val Val Arg

100 105 110

Ile Val Thr Pro Gly Thr Ile Cys Asp Glu Ala Leu Leu Gln Glu Arg

115 120 125

Gln Asp Asn Leu Leu Ala Ala Ile Trp Gln Asp Ser Lys Gly Phe Gly

130 135 140

Tyr Ala Thr Leu Asp Ile Cys Ser Gly Arg Phe Arg Cys Ser Glu Pro

145 150 155 160

Ala Asp Arg Glu Thr Met Ala Ala Glu Leu Gln Arg Thr Asn Pro Ala

165 170 175

Glu Leu Leu Tyr Ala Glu Asp Phe Ala Glu Met Ser Leu Ile Glu Gly

180 185 190

Arg Arg Gly Leu Arg Arg Arg Pro Leu Trp Glu Phe Glu Ile Asp Thr

195 200 205

Ala Arg Gln Gln Leu Asn Leu Gln Phe Gly Thr Arg Asp Leu Val Gly

210 215 220

Phe Gly Val Glu Asn Ala Pro Arg Cys Leu Cys Ala Ala Gly Cys Leu

225 230 235 240

Leu Gln Tyr Ala Lys Asp Thr Gln Arg Thr Thr Leu Pro His Ile Arg

245 250 255

Ser Ile Thr Met Glu Arg Glu Gln Asp Ser Ile Ile Met Asp Ala Ala

260 265 270

Thr Arg Arg Asn Leu Glu Ile Thr Gln Asn Leu Ala Gly Gly Ala Glu

275 280 285

Asn Thr Leu Ala Ser Val Leu Asp Cys Thr Val Thr Pro Met Gly Ser

290 295 300

Arg Met Leu Lys Arg Trp Leu His Met Pro Val Arg Asp Thr Arg Val

305 310 315 320

Leu Leu Glu Arg Gln Gln Thr Ile Gly Ala Leu Gln Asp Phe Thr Ala

325 330 335

Gly Leu Gln Pro Val Leu Arg Gln Val Gly Asp Leu Glu Arg Ile Leu

340 345 350

Ala Arg Leu Ala Leu Arg Thr Ala Arg Pro Arg Asp Leu Ala Arg Met

355 360 365

Arg His Ala Phe Gln Gln Leu Pro Glu Leu Arg Ala Gln Leu Glu Thr

370 375 380

Val Asp Ser Ala Pro Val Gln Ala Leu Arg Glu Lys Met Gly Glu Phe

385 390 395 400

Ala Glu Leu Arg Asp Leu Leu Glu Arg Ala Ile Ile Asp Thr Pro Pro

405 410 415

Val Leu Val Arg Asp Gly Gly Val Ile Ala Ser Gly Tyr Asn Glu Glu

420 425 430

Leu Asp Glu Trp Arg Ala Leu Ala Asp Gly Ala Thr Asp Tyr Leu Glu

435 440 445

Arg Leu Cys Val Arg Glu Arg Glu Arg Thr Gly Leu Asp Thr Leu Lys

450 455 460

Cys Gly Phe Asn Ala Val His Gly Tyr Tyr Ile Gln Ile Ser Arg Gly

465 470 475 480

Gln Ser His Leu Ala Pro Ile Asn Tyr Met Arg Arg Gln Thr Leu Lys

485 490 495

Asn Ala Glu Arg Tyr Ile Ile Pro Glu Leu Lys Glu Tyr Glu Asp Lys

500 505 510

Val Leu Thr Ser Lys Gly Lys Ala Leu Ala Leu Glu Lys Gln Leu Tyr

515 520 525

Glu Glu Leu Phe Asp Leu Leu Leu Pro His Leu Glu Ala Leu Gln Gln

530 535 540

Ser Ala Ser Ala Leu Ala Glu Leu Asp Val Leu Val Asn Leu Ala Glu

545 550 555 560

Arg Ala Tyr Thr Leu Asn Tyr Thr Cys Pro Thr Phe Ile Asp Lys Pro

565 570 575

Gly Ile Arg Ile Thr Glu Gly Arg His Pro Val Val Glu Gln Val Leu

580 585 590

Asn Glu Pro Phe Ile Ala Asn Pro Leu Asn Leu Ser Pro Gln Arg Arg

595 600 605

Cys Leu Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Thr Tyr Met

610 615 620

Arg Gln Thr Ala Leu Ile Ala Leu Met Ala Tyr Ile Gly Ser Tyr Val

625 630 635 640

Pro Ala Gln Lys Val Glu Ile Gly Pro Ile Asp Arg Ile Phe Thr Arg

645 650 655

Val Gly Ala Ala Asp Asp Leu Ala Ser Gly Arg Ser Thr Phe Met Val

660 665 670

Glu Met Thr Glu Thr Ala Asn Ile Leu His Asn Ala Thr Glu Tyr Ser

675 680 685

Leu Val Leu Met Asp Glu Ile Gly Arg Gly Thr Ser Thr Tyr Asp Gly

690 695 700

Leu Ser Leu Ala Trp Ala Cys Ala Glu Asn Leu Ala Asn Lys Ile Lys

705 710 715 720

Ala Leu Cys Leu Phe Ala Thr His Tyr Phe Glu Leu Thr Gln Leu Pro

725 730 735

Glu Lys Met Glu Gly Val Ala Asn Val His Leu Asp Ala Leu Glu His

740 745 750

Gly Asp Thr Ile Ala Phe Met His Ser Val Gln Asp Gly Ala Ala Ser

755 760 765

Lys Ser Tyr Gly Leu Ala Val Ala Ala Leu Ala Gly Val Pro Lys Glu

770 775 780

Val Ile Lys Arg Ala Arg Gln Lys Leu Arg Glu Leu Glu Ser Ile Ser

785 790 795 800

Pro Asn Ala Ala Ala Thr Gln Val Asp Gly Thr Gln Met Ser Leu Leu

805 810 815

Ser Val Pro Glu Glu Thr Ser Pro Ala Val Glu Ala Leu Glu Asn Leu

820 825 830

Asp Pro Asp Ser Leu Thr Pro Arg Gln Ala Leu Glu Trp Ile Tyr Arg

835 840 845

Leu Lys Ser Leu Val

850

Claims (11)

1. A mismatch binding protein, characterized in that, the amino acid sequence of the mismatch binding protein is selected from the group: 1) The amino acid sequence obtained by mutating the 120th and 151st serines in the amino acid sequence shown in SEQ ID NO:52 to cysteine; 2) The amino acid sequence obtained by mutating the 157th leucine and the 233rd glycine into cysteine in the amino acid sequence shown in SEQ ID NO:52; or 3) The serine at the 120th position, the leucine at the 157th position, the glutamic acid at the 451st position and the methionine at the 609th position in the amino acid sequence shown in SEQ ID NO:52 are all mutated into cysteine, and the obtained amino acid sequence. 2. The mismatch binding protein according to claim 1, wherein the amino acid sequence of the mismatch binding protein is as shown in SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO:62. 3. A gene encoding the mismatch binding protein of claim 1. 4. The coding gene according to claim 3, wherein the sequence of the gene is shown in SEQ ID NO:53, SEQ ID NO:55 or SEQ ID NO:61. 5. A vector comprising the coding gene of claim 3. 6. A host cell, characterized in that the host cell contains the coding gene of claim 3. 7. Use of the host cell according to claim 6 in the production of a mismatch binding protein. 8. A method for preparing a mismatch binding protein, characterized in that the method comprises the following steps: a. culturing the host cell of claim 6 to produce said mismatch binding protein; and b. Isolating the mismatch binding protein from the culture medium. 9. The use of the mismatch binding protein of claim 1 in identifying and binding DNA mismatches. 10. The application according to claim 9, wherein the mismatch binding protein is used in a free liquid state to identify and bind mismatched DNA, or is used in combination with EGFP and CBM to immobilize the mismatch binding protein on the column Used to recognize and bind mismatched DNA. 11. A method for transforming a wild-type mismatch binding protein to improve thermal stability, characterized in that the method comprises the following steps: a. comparing the amino acid sequence of the wild-type mismatch binding protein with the amino acid sequence shown in SEQ ID NO:52; b. Transform the coding sequence of the wild-type mismatch binding protein so that the mutation corresponding to the amino acid sequence shown in SEQ ID NO:52 in the coded amino acid sequence is selected from one of the following groups: 1) Both serine at position 120 and position 151 are mutated to cysteine, 2) Both leucine at position 157 and glycine at position 233 are mutated to cysteine, or 3) Serine at position 120, leucine at position 157, glutamic acid at position 451 and methionine at position 609 are all mutated to cysteine; c. Directly transfecting the coding sequence obtained in b into a suitable host cell or introducing a vector into a suitable host cell; d. culturing the host cells obtained in c; e. isolating the mismatch binding protein produced by the host cell from the culture system obtained in step d; and f. Determining the thermal stability of the mismatch binding protein.

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