CN116574710A

CN116574710A - DNA polymerase with strand displacement function and application thereof

Info

Publication number: CN116574710A
Application number: CN202310441580.7A
Authority: CN
Inventors: 逯晓云; 祁雪荣
Original assignee: Tianjin Zhonghe Gene Technology Co ltd
Current assignee: Tianjin Zhonghe Gene Technology Co ltd
Priority date: 2023-04-23
Filing date: 2023-04-23
Publication date: 2023-08-11

Abstract

The application discloses a DNA polymerase with a strand displacement function and application thereof, belonging to the field of genetic engineering. In order to increase the diversity of the polymerase with strand displacement function to increase the efficiency or applicability of rolling circle amplification, the present application provides a DNA polymerase selected from the group consisting of proteins whose amino acid sequence is from 21 st to 604 th in sequence 1 or whose amino acid sequence is from 21 st to 596 th in sequence 2 or whose amino acid sequence is from 21 st to 615 th in sequence 3 or whose amino acid sequence is from 21 st to 606 th in sequence 4 or whose amino acid sequence is from 1 or whose amino acid sequence is from 2 nd or whose amino acid sequence is from 3 nd or whose amino acid sequence is from 4. The application also provides application of the DNA polymerase in DNA rolling circle amplification. The DNA polymerase provided by the application can still realize the signal amplification effect under the condition of extremely low template concentration, and can be widely applied to DNA amplification, nucleic acid molecule sequencing and biomedical diagnosis.

Description

DNA polymerase with strand displacement function and application thereof

Technical Field

The application belongs to the technical field of genetic engineering, and particularly relates to a DNA polymerase with a strand displacement function and application thereof.

Background

The strand displacement amplification technology, also called rolling circle amplification technology (Rolling circle amplification, RCA), is derived from the replication of pathogenic microorganisms in nature, and uses a DNA polymerase with strand displacement function, a modified primer and deoxyribonucleoside triphosphates to perform the replication process using circular DNA as a template. The technology is characterized in that the technology does not need a complex temperature raising and lowering procedure, does not limit the requirements of dense instruments, and has good specificity and high sensitivity.

The traditional method for amplifying DNA in large quantity is based on cell culture, has complicated flow and higher cost, and can prepare the finished product DNA through multi-step purification if large-scale escherichia coli culture is needed to be completed in a fermentation tank, then target DNA is released by splitting bacteria. Compared with other amplification technologies, the RCA technology has the greatest advantage that isothermal amplification can be realized without a complex primer design process, and an amplification system is greatly simplified. Under the condition of extremely low template concentration, the signal amplification effect can still be well realized, and the method is widely applied to DNA amplification, nucleic acid molecule sequencing and biomedical diagnosis.

Disclosure of Invention

The application aims to solve the technical problems that: how to increase the diversity of the polymerase with strand displacement function to increase the efficiency or applicability of rolling circle amplification.

To solve the above technical problem, the present application provides, in a first aspect, a protein selected from any one of the following:

a1 Any one of the proteins of SEQ ID No.1-4, the protein of which the amino acid sequence is the 21 st to 604 th positions of SEQ ID No.1, the protein of which the amino acid sequence is the 21 st to 596 th positions of SEQ ID No.2, the protein of which the amino acid sequence is the 21 st to 615 th positions of SEQ ID No.3 or/and the protein of which the amino acid sequence is the 21 st to 606 th positions of SEQ ID No. 4;

a2 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A1).

Protein the above-mentioned protein can be artificially synthesized, or can be obtained by synthesizing its coding gene first and then making biological expression.

The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.

In a second aspect the present application provides a biomaterial associated with a protein as described above, said biomaterial being selected from any one of the following:

b1 A nucleic acid molecule encoding the above protein;

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

b3 A recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

Further, in the biological material, the nucleic acid molecule of B1) is selected from any one of the following:

g1 A DNA molecule with the coding sequence of the coding strand shown in SEQ ID No. 5;

g2 A DNA molecule with the coding sequence of the coding strand shown in SEQ ID No. 6;

g3 A DNA molecule with the coding sequence of the coding strand shown in SEQ ID No. 7;

g4 A DNA molecule with the coding sequence of the coding strand shown in SEQ ID No. 8;

g5 A DNA molecule which has 80% or more identity to the DNA molecule defined in g 1) to g 4) and which encodes the above protein.

In the present application, identity refers to identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

Further, in the above biological material, the expression cassette of B2) refers to a DNA molecule capable of expressing the above protein in a host cell, and the DNA molecule may include not only a promoter for promoting transcription of a gene encoding the protein but also a terminator for terminating transcription of the protein gene.

Further, in the above biological material, the expression cassette of B2) may further include an enhancer sequence. Promoters useful in the present application include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters and inducible promoters.

Further, in the above-mentioned related biological material, the recombinant vector of B3) may contain a DNA molecule for encoding a protein shown in any one or more of SEQ ID Nos. 5 to 8. Recombinant vectors containing the protein-encoding gene expression cassettes can be constructed using microbial expression vectors.

Further, in the above-mentioned related biological material, the recombinant microorganism of B4) may be specifically yeast, bacteria, algae and fungi.

In a third aspect the application provides the use of a protein as described above in any one of the following:

c1 Use in DNA amplification);

c2 For the preparation of a DNA amplified product;

c3 Use as a DNA polymerase;

c4 For the preparation of DNA polymerase preparations).

In a fourth aspect the present application provides the use of a biomaterial as described above in any one of the following:

d1 Use in DNA amplification);

d2 For the preparation of a DNA amplified product;

d3 Use in the preparation of DNA polymerase;

d4 For the preparation of DNA polymerase preparations).

Further, in the above application, the DNA amplification may be DNA rolling circle amplification.

Further, in the application, the product is a reagent or a kit, and the reagent or the kit can be used for DNA amplification.

In a fifth aspect the application provides a reagent or kit comprising a protein as described above or/and a biological material as described above.

In a sixth aspect the present application provides a method of DNA amplification comprising subjecting a protein as described above to DNA amplification.

Further, in the method, the DNA amplification may be rolling circle amplification.

In yet another embodiment of the present application, the additional reaction system for DNA amplification comprises: more than 1ng of double-stranded circular DNA template, reaction Buffer10×, dNTP, inorganic pyrophosphatase, DNA polymerase with strand displacement function (the above protein), DNase-free Water.

The reaction conditions are as follows:

(1) Uniformly mixing 2ng of double-stranded circular DNA template, 6nt random primer ((final concentration 10 mu M) and reaction Buffer 10X, and cooling after denaturation for 3min at 95 ℃ in a metal bath;

(2) dNTPs (1 mM final concentration) and DNA polymerase having a strand displacement function (the above protein) were added thereto, and DNase-free Water was added to 50. Mu.L and reacted at 30℃overnight to obtain an amplified product containing the desired fragment.

Further, in the method, the template for DNA amplification may be a double-stranded DNA molecule or a single-stranded DNA molecule.

Further, in the method, the amount of the DNA polymerase having a strand displacement function (the above protein) added in the reaction system for DNA amplification may be 0.1 to 10ng.

Further, in the method, the length of the primer in the reaction system for DNA amplification may be 6-15nt.

In an embodiment of the present application, at least one pyrophosphatase enzyme is employed which has the function of catalyzing the conversion of one molecule of pyrophosphate to two molecules of phosphate ions, preferably an inorganic pyrophosphatase enzyme.

In embodiments of the application, the nucleotides employed are one or more nucleotide mixtures. The nucleotide refers to one or more of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP) and derivatives thereof. The nucleotides, preferably one or more of adenine (a), guanine (G), thymine (T), cytosine (C), may be provided in the form of one or more suitable mixtures. Two, three, four, preferably four nucleotides (A, G, T and C) are used in the method of synthesizing DNA.

In embodiments of the present application, the metal ions employed may contain at least one metal ion, which may be a salt of a divalent metal ion: magnesium (Mg) ²⁺ ) Manganese (Mn) ²⁺ ) Calcium (Ca) ²⁺ ) Etc., or salts of monovalent metal ions: lithium (Li) ⁺ ) Sodium (Na) ⁺ ) Or potassium (K) ⁺ ). Preferably sodium (Na ⁺ ) And potassium (K) ⁺ ) But is not limited thereto.

The application breaks through the strategy of developing new enzymes based on the mode microorganism genome information by the traditional homology library construction comparison analysis, utilizes alpha fold2 to analyze proteins with homology of more than 30 percent and perform structural simulation, integrates structural information, analysis kinetic information and enzyme function verification, obtains DNA polymerase with higher efficiency and better performance and having a strand displacement function, namely the DNA polymerase for amplifying nucleic acid molecules based on the rolling circle amplification technology, and provides a method for solving the problem of amplification of a large number of nucleic acid molecules.

The strategy has the advantages that the strategy for mining the new enzyme according to the mode microorganism genome information is different from the previous strategy by analyzing the metagenome sequences of the environmental microorganism populations such as industrial wastewater, fermented beverages, biogas power plants, plant lodging populations and the like and mining the new enzyme.

Another strategy of the present application is advantageous in that the entire sequence annotated as DNA polymerase in the metagenome is subjected to a library-building analysis, and through multiple sequence comparison, a sequence library-building analysis with a similarity to the DNA polymerase group B sequence higher than 30% is screened, which has a larger difference from the similarity to the sequence of 90% commonly used in the traditional new enzyme mining and has a wider coverage rate.

Another strategy of the present application is advantageous in that protein sequences with more than 30% homology are analyzed by using alpha fold2 and structural simulation is performed, and proteins with potential strand displacement activity are screened according to structural information and analysis results of analysis kinetic information.

The beneficial technical effects obtained by the application are as follows:

1) Providing 4 DNA polymerases with strand displacement amplification activity provides a method for amplifying nucleic acid molecules.

2) Under the condition of extremely low template concentration, the DNA polymerase or the reagent or the kit can still realize the signal amplification function well, and can be widely applied to DNA amplification, nucleic acid molecule sequencing and biomedical diagnosis.

Drawings

FIG. 1 is a schematic diagram of the rolling circle amplification method of the present application. After high-temperature denaturation, the double-stranded circular DNA is subjected to base pairing with a modified primer, DNA polymerase with a strand displacement effect simultaneously starts to replicate at a plurality of sites of a template, DNA is synthesized along the DNA template, the complementary strand of the template is simultaneously displaced, and the displaced complementary strand becomes a new template for amplification. Grey is the DNA polymerase with strand displacement function of the present application, and extends from 5 'to 3'.

FIG. 2 is a diagram of a purified protein gel of the DNA polymerase of the present application.

FIG. 3 is a diagram of electrophoresis of nucleic acids after a rolling circle amplification reaction of DNA polymerase. Lanes 1 are 1kb Marker, lanes 2, 3 are added with microbial DNA polymerase from industrial wastewater, lanes 4, 5 are added with microbial DNA polymerase from fermented beverage, lanes 6, 7 are added with microbial DNA polymerase from biogas power plant, lanes 8, 9 are added with microbial DNA polymerase from boarder race, and lanes 10, 11 are amplification negative and positive controls.

FIG. 4 is a diagram showing the electrophoresis of nucleic acid after the rolling circle amplification of single-stranded DNA by the DNA polymerase of the present application is reacted by adding DNA templates of different concentrations respectively. Wherein, lane 1 is a 1kb Marker, lane 2 is a reaction result of rolling circle amplification by throwing 0.1. Mu.g of single-stranded DNA, lane 3 is a reaction result of rolling circle amplification by throwing 2. Mu.g of single-stranded DNA, lane 4 is a reaction result of rolling circle amplification by throwing 5. Mu.g of single-stranded DNA, and lane 5 is a reaction result of rolling circle amplification by throwing 10. Mu.g of single-stranded DNA.

FIG. 5 shows the electrophoresis of double-stranded DNA amplified by rolling circle with DNA polymerase, after the addition of thio-modified primers of different lengths, respectively. Wherein, lane 1 is a 1kb Marker, lanes 2 and 3 are the reaction results of rolling circle amplification by adding 15nt of the thio-modified primer, lanes 4 and 5 are the reaction results of rolling circle amplification by adding 8nt of the thio-modified primer, and lanes 6 and 7 are the reaction results of rolling circle amplification by adding 6nt of the thio-modified primer.

FIG. 6 shows the results of sequencing validation of the products obtained from the DNA polymerase rolling circle amplification reaction.

Detailed Description

The action principle and the process of the method are introduced as follows:

firstly, uniformly mixing a modified primer and double-stranded circular DNA, heating for denaturation, then placing on ice, finally adding mixed deoxyribonucleoside triphosphates into a reaction sample to trigger a linear RCA reaction, generating a long and continuous repeated DNA single strand complementary to the circular DNA, and performing a second round of amplification reaction under the action of DNA polymerase with a strand displacement function. And so on, long double-stranded DNA products are formed after multiple rounds of the above reactions. The method has high sensitivity, can be widely applied to DNA amplification, nucleic acid sequencing and biomedical diagnosis, and can achieve the expected effect.

FIG. 1 is a schematic diagram of the rolling circle amplification method of the present application. After high-temperature denaturation, the double-stranded circular DNA is subjected to base pairing with a modified primer, DNA polymerase with a strand displacement effect simultaneously starts to replicate at a plurality of sites of a template, DNA is synthesized along the DNA template, the complementary strand of the template is simultaneously displaced, and the displaced complementary strand becomes a new template for amplification. Filled ellipses represent DNA polymerases of the present application with strand displacement function, extending in a direction from 5 'to 3'.

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

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the following examples, vector pET28a (+) was purchased from MiaoLingBio cat: pET-28a-CCNK (human).

In the examples below, JM109 (DE 3) was purchased from Weidi, cat: JM109.

In the following examples, the composition in the 2 XYT medium is: 1.6% tryptone, 1% yeast extract, 0.5% NaCl, the remainder being water.

In the following examples, the composition of Buffer A solution was: 20mM Tris-HCl,500mM NaCl,20mM lmidazole,5% (v/v) Glycerol, the remainder being water, the pH of the solution was 7.4.

In the following examples, the composition of Buffer B eluate was: 20mM Tris-HCl,500mM NaCl,500mM lmidazole,5% (v/v) Glycerol, the remainder being water, the pH of the solution was 7.4.

In the following examples, the composition of Buffer C is: 50mM Tris-HCl,100mM KCl,1mM DTT,50% (v/v) Glycerol, the remainder being water, the pH of the solution was 7.4.

The reaction Buffer10 x composition was: 500mM Tris-HCl (pH 7.5, 25 ℃ C.), 100mM MgCl ₂ ，100mM(NH ₄ ) ₂ SO ₄ 40mM DTT (dithiothreitol) with the remainder being water.

The 6nt random primer (0.1 mM) in the following examples was purchased from Bai Lai Bo, cat: BTN120679.

dNTPs in the following examples refer to a mixture of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), wherein the ratio of the amounts of dATP, dGTP, dCTP and dTTP is 1:1:1:1, dNTPs are purchased from ABclonal: RK20120.

The composition of reaction Buffer10 x in the examples below is: 500mM Tris-HCl (pH 7.5, 25 ℃ C.), 100mM MgCl ₂ ，100mM(NH ₄ ) ₂ SO ₄ 40mM DTT (dithiothreitol) with the remainder being water.

In the following examples, the Marker in the nucleic acid gel electrophoresis pattern was 1kb Marker (DL 10004), and the bands were 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000 and 12000bp from bottom to top, respectively.

Example 1 selection of DNA polymerase having strand displacement function

The metagenome sequence of the environmental organism microorganism population such as industrial wastewater, fermented beverage, biogas power plant, plant lodging population and the like is analyzed and new enzymes are excavated. All sequences annotated as DNA polymerase in the metagenome were subjected to a library construction analysis, and sequence library construction analysis with a similarity to the DNA polymerase group B sequence of more than 30% was selected by multiple sequence alignment. And analyzing the protein sequence with the homology of more than 30% by using alpha fold2, performing structural simulation, and screening the protein with potential strand displacement activity according to structural information and analysis results of analysis kinetic information. Through a large number of screening and verification, 4 DNA polymerases with strand displacement amplification activities are finally obtained.

Firstly, unipro is utilized to find the protein sequence of Phi29DNA polymerase, the protein sequence takes the fasta format amino acid sequence as input, the database construction analysis is carried out on all sequences annotated as DNA polymerase in a metagenome, and the strategy screening of mining new enzymes according to the mode microbial genome information is distinguished from the prior strategy screening of mining new enzymes according to the mode microbial genome information by analyzing metagenome sequences of environmental biological microbial populations such as industrial wastewater, fermented beverage, biogas power plants, plant lodging populations and the like, wherein the sequence database construction analysis with the similarity higher than 30 percent has larger difference and wider coverage than the sequence similarity of 90 percent commonly used in the traditional new enzyme mining. And carrying out homologous sequence search on the protein according to the analysis result, and carrying out alpha fold2 modeling analysis on the protein with the homology of more than 30%. And generating a parameter file by using a rosetta program, performing molecular dynamics simulation, further deepening the reliability of a protein sequence, and finally screening out the DNA polymerase with the strand displacement function activity and performing a test to verify the activity of the DNA polymerase.

The 4 DNA polymerases obtained by the preliminary screening were designated as DNA polymerase 1, DNA polymerase 2, DNA polymerase 3 and DNA polymerase 4, respectively. Wherein the DNA polymerase 1 is from industrial wastewater microbial assembly of ethylene phthalate, metagenome ID: ERZ650227. The amino acid sequence is SEQ ID No.1 at positions 21-604, and the nucleotide sequence of the encoding gene is SEQ ID No.5.DNA polymerase 2 is from fermented beverage microorganisms in food production, metagenomic ID: ERZ724034. The amino acid sequence is SEQ ID No.2 at positions 21-596, and the nucleotide sequence of the encoding gene is SEQ ID No.6.DNA polymerase 3 is from a microorganism of a biogas power plant, metagenomic ID: ERZ773357. The amino acid sequence is SEQ ID No.3 at positions 21-615, and the nucleotide sequence of the encoding gene is SEQ ID No.7.DNA polymerase 4 is from a microorganism hosting race, metagenome ID: ERZ781244. The amino acid sequence is SEQ ID No.4 at positions 21-606, and the nucleotide sequence of the encoding gene is SEQ ID No.8.

TABLE 1 amino acid and nucleotide sequences of 4 DNA polymerases

Example 2 expression and purification of DNA polymerase having strand displacement function

(1) And respectively constructing target sequences on pET28a (+) vectors to obtain recombinant expression vectors, and respectively transforming JM109 (DE 3) competence by the recombinant expression vectors to obtain recombinant bacteria.

The specific steps for constructing the target sequence into the pET28a (+) vector are as follows:

taking a recombinant expression vector for expressing DNA polymerase 1 as an example, replacing a fragment between NdeI and Xhol cleavage recognition sites of the pET28a (+) vector (a small fragment between NdeI and Xhol cleavage recognition sites) with a DNA molecule shown in SEQ ID No.5, and keeping other nucleotide sequences of the pET28a (+) vector unchanged to obtain a recombinant expression vector pET28a/DNA pol-1. The expressible amino acid sequence of the recombinant expression vector pET28a/DNA pol-1 is the fusion protein of SEQ ID No.1, wherein the 21 st to 604 th positions of the SEQ ID No.1 are the amino acid sequence of DNA polymerase 1.

The construction method of the recombinant expression vector pET28a/DNA pol-2 refers to the recombinant expression vector pET28a/DNA pol-1, and the only difference is that: the DNA molecule constructed to the pET28a (+) vector is replaced by a DNA molecule with a nucleotide sequence shown as SEQ ID No.6, and the rest operation is kept unchanged to obtain a recombinant expression vector pET28a/DNA pol-2. The expressible amino acid sequence of the recombinant expression vector pET28a/DNA pol-2 is the fusion protein of SEQ ID No.2, wherein the 21 st to 596 th positions of the SEQ ID No.2 are the amino acid sequence of DNA polymerase 2.

The construction method of the recombinant expression vector pET28a/DNA pol-3 refers to the recombinant expression vector pET28a/DNA pol-1, and the only difference is that: the DNA molecule constructed to the pET28a (+) vector was replaced with a DNA molecule with the nucleotide sequence shown in SEQ ID No.7, and the remaining operations remained unchanged to obtain a recombinant expression vector pET28a/DNA pol-3. The expressible amino acid sequence of the recombinant expression vector pET28a/DNA pol-3 is the fusion protein of SEQ ID No.3, wherein the 21 st to 615 th positions of SEQ ID No.3 are the amino acid sequence of DNA polymerase 3.

The construction method of the recombinant expression vector pET28a/DNApol-4 refers to the recombinant expression vector pET28a/DNA pol-1, and only differs in that: the DNA molecule constructed to the pET28a (+) vector was replaced with a DNA molecule with the nucleotide sequence shown in SEQ ID No.8, and the remaining operations remained unchanged to obtain a recombinant expression vector pET28a/DNA pol-4. The expressible amino acid sequence of the recombinant expression vector pET28a/DNA pol-4 is the fusion protein of SEQ ID No.4, wherein the 21 st-606 th positions of the SEQ ID No.4 are the amino acid sequence of DNA polymerase 4.

(2) Recombinant bacteria were inoculated into 4mL of kan-resistant (10 mg/L) 2 XYT medium, cultured overnight at 37℃and positive clones were selected.

Wherein the positive clones were designated JM109 (DE 3)/pET 28a/DNA pol-1, JM109 (DE 3)/pET 28a/DNA pol-2, JM109 (DE 3)/pET 28a/DNA pol-3 and JM109 (DE 3)/pET 28a/DNA pol-4, respectively.

(3) After the completion of the step (2), the above bacterial liquid was transferred to a kan (10 mg/L) resistant 2 XYT medium in 1/200 volume, and shake-cultured at 37℃at 220rpm to OD ₆₀₀ =about 0.6 (0.4 to 0.8 are all possible).

(4) After the completion of the step (3), 0.1mM IPTG was added to the bacterial solutions of JM109 (DE 3)/pET 28a/DNA pol-1, JM109 (DE 3)/pET 28a/DNA pol-2, JM109 (DE 3)/pET 28a/DNA pol-3 and JM109 (DE 3)/pET 28a/DNA pol-4, respectively, and the culture was carried out at 16℃for 12-14 hours with shaking at 220 rpm.

(5) After the completion of the step (4), the cells were collected by centrifugation at 5500rpm at 4℃for 8 min.

(6) After the step (5) is completed, the mixture is evenly mixed by shaking by adopting a Buffer A solution, then the mixture is placed on ice, the thalli are crushed by using a high-pressure crusher, and then the thalli are centrifuged for 40min at 4 ℃ and 10000rpm, and the supernatants are respectively collected.

(7) And (3) respectively purifying the supernatant obtained in the step (6) by adopting nickel column affinity chromatography. Taking the purification of a fusion protein (or a fusion protein comprising DNA polymerase 1) whose amino acid sequence is the sequence SEQ ID NO.1 as an example, the specific steps are as follows:

the nickel column (HYCX, cat# SA 004050) was rinsed twice with 20 column volumes of pure water, and then rinsed with 10 column volumes of Buffer A solution. Then loading samples, wherein the concrete operation is as follows: and (3) slowly passing the supernatant obtained in the step (6) through a Ni affinity chromatography column at a flow rate of 0.5mL/min, and repeating the steps once again. Then eluting with 15 column volumes of eluent and collecting the liquid after passing through the column with target protein, wherein the eluent consists of Buffer A and Buffer B, the volume fraction of Buffer B in the eluting process is increased from 0% to 100%, and the volume fraction of corresponding Buffer A is decreased from 100% to 0%.

(8) Finally, ultrafiltration is carried out by using an ultrafiltration tube, and the specific steps are as follows: and replacing the solution containing the target protein with Buffer C to obtain the concentrated and purified target protein. The protein is diluted to 1mg/ml, the gel electrophoresis pattern is shown in figure 2, and the target protein of 68kDa is obtained after concentration.

The steps of homozygosity and concentration of the remaining 3 fusion proteins are referred to (7) and (8), and the remaining steps remain unchanged.

Example 3 Rolling circle amplification of DNA polymerase with Strand Displacement function

3.1 double-stranded DNA Rolling circle amplification with Strand Displacement function DNA polymerase

The fusion proteins (or recombinant DNA polymerases) shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4, respectively, were used for double-stranded DNA rolling circle amplification.

The reaction system is as follows:

more than 1ng of double-stranded circular DNA template, reaction Buffer10×, dNTP, inorganic pyrophosphatase, DNA polymerase with strand displacement function, DNase-free Water. Wherein the nucleotide sequence of the double-stranded circular DNA template is SEQ ID No.9.

And simultaneously setting a negative control and a positive control, wherein the DNA polymerase with the strand displacement function of the reaction system is replaced by water in the negative control. The positive control was that the DNA polymerase having the strand displacement function of the above reaction system was replaced with Phi29DNA polymerase (purchased in Norfluaz, cat# N106-02).

The reaction conditions are as follows:

(3) Uniformly mixing 2ng of double-stranded circular DNA template, 6nt random primer ((final concentration 10 mu M) and reaction Buffer 10X, and cooling after denaturation for 3min at 95 ℃ in a metal bath;

(4) dNTPs (1 mM final concentration) were added, and DNA polymerase having a strand displacement function was added thereto, and DNase-free Water was added to 50. Mu.L, followed by reaction at 30℃for 16 hours overnight to obtain an amplified product containing the target fragment.

(5) The amplified product of DNA polymerase 1 was subjected to second generation pool sequencing as shown in fig. 6, region one: nucleotide sequence of partial DNA template, region two: sequencing results after partial segmentation, region three: single base amplification accuracy results. Therefore, the accuracy of the amplified product of the DNA polymerase with the strand displacement function is more than 99%, and the isothermal amplification effect is realized.

And (3) analyzing and identifying the amplified product by utilizing gel electrophoresis, wherein the electrophoresis condition is that the voltage is 130V, and the electrophoresis time is 30min. And carrying out qualitative analysis on the product according to the brightness of the electrophoresis band.

The results are shown in FIG. 3, wherein lanes 1 are 1kb Marker, lanes 2, 3 are products obtained by adding DNA polymerase 1, lanes 4, 5 are products obtained by adding DNA polymerase 2, lanes 6, 7 are products obtained by adding DNA polymerase 3, lanes 8, 9 are products obtained by DNA polymerase 4, and lanes 10, 11 are negative and positive controls, respectively.

The results of fig. 3 show that: the DNA polymerase 1, the DNA polymerase 2, the DNA polymerase 3 and the DNA polymerase 4 (or recombinant DNA polymerase) with the strand displacement function, which are shown by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, can be used for double-stranded DNA rolling circle amplification, which shows that the 4 DNA polymerases are all DNA polymerases with the strand displacement function.

3.2 Single-stranded DNA Rolling circle amplification with Strand Displacement function DNA polymerase

Example 2 the prepared fusion protein (or recombinant DNA polymerase) with the amino acid sequence of SEQ ID No.1, respectively, was used for double-stranded DNA rolling circle amplification.

The reaction system is as follows:

more than 1ng of single-stranded circular DNA template, reaction Buffer10×, 6nt random primer (0.1 mM), dNTP, inorganic pyrophosphatase, DNA polymerase with strand displacement function, DNase-free Water. Wherein the nucleotide sequence of the single stranded circular DNA template is SEQ ID No.10.

The reaction conditions are as follows: the single-stranded circular DNA templates (0.1 ng, 2ng, 5ng, 10ng, etc.), hydrolysis primers (6 nt random primer, final concentration 10. Mu.M) were mixed uniformly at 10X in the reaction Buffer, dNTPs (final concentration 1 mM) and DNA polymerase 1 having a strand displacement function were added thereto, and DNase-free Water was added to 50. Mu.L and reacted overnight at 30℃for 16 hours to obtain an amplified product containing the target fragment.

The results are shown in FIG. 4, and FIG. 4 is a diagram of nucleic acid electrophoresis after the rolling circle amplification of single-stranded DNA by the DNA polymerase of the present application. Wherein, lane 1 is a 1kb Marker, lane 2 is a reaction result of rolling circle amplification by throwing 0.1ng of single-stranded DNA template, lane 3 is a reaction result of rolling circle amplification by throwing 2ng of single-stranded DNA template, lane 4 is a reaction result of rolling circle amplification by throwing 5ng of single-stranded DNA template, and lane 5 is a reaction result of rolling circle amplification by throwing 10ng of single-stranded DNA template.

The results of fig. 4 show that: the reaction system and reaction conditions described above can form a large amount of long fragment nucleic acid products, confirming that the DNA polymerase has strand displacement function in the range of 0.1ng to 10ng, preferably in the range of 0.5ng to 5ng, more preferably in the range of 2ng to 5ng of single-stranded or double-stranded circular DNA substrate concentration.

3.3, respectively adding thio-modified primers with different lengths into DNA polymerase (or recombinant DNA polymerase) with a strand displacement function and an amino acid sequence shown as SEQ ID No.1 for rolling and replacing amplification

The reaction system is as follows:

more than 1ng of single-stranded circular DNA was used as a template, reaction Buffer10×, 6nt random primer (0.1 mM), dNTP, inorganic pyrophosphatase, DNA polymerase with strand displacement function, DNase-free Water. Wherein the nucleotide sequence of the single stranded circular DNA template is SEQ ID No.10.

The reaction conditions are as follows:

(2) Deoxyribonucleoside triphosphates (final concentration: 1 mM) were added, DNA polymerase having a strand displacement function was added, DNase-free Water was added to 50. Mu.L, and the mixture was reacted at 30℃overnight to obtain an amplified product containing the desired fragment.

As a result, FIG. 5 shows the result of the rolling circle amplification of double-stranded DNA by the DNA polymerase of the present application, and the electrophoresis of the nucleic acid after the reaction of adding thio-modified primers of different lengths, respectively. In which lane 1 is a 1kb Marker. Lanes 2 and 3 show the results of the rolling circle amplification reaction in which 15nt of the thio-modified primer was introduced, lanes 4 and 5 show the results of the rolling circle amplification reaction in which 8nt of the thio-modified primer was introduced, and lanes 6 and 7 show the results of the rolling circle amplification reaction in which 6nt of the thio-modified primer was introduced.

The results of fig. 5 show that: a large number of long fragment nucleic acid products can be formed under the reaction system and the reaction conditions, and the primer adopted by the verified DNA polymerase with the strand displacement function is proved to be a degradation-resistant modified primer, and the modification mode is preferably thio modification. The primer length is preferably 6 to 15nt, more preferably 6 to 8nt.

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

Claims

1. A protein characterized in that: the protein is selected from any one of the following:

2. A biological material associated with the protein of claim 1, characterized in that: the biological material is selected from any one of the following:

b1 A nucleic acid molecule encoding the protein of claim 1;

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

3. The biomaterial according to claim 2, characterized in that: b1 A nucleic acid molecule selected from any one of the following:

g5 A DNA molecule having 80% or more identity to the DNA molecule defined in g 1) -g 4) and encoding the protein of claim 1.

4. Use of the protein of claim 1 in any of the following:

c1 Use in DNA amplification);

c2 For the preparation of a DNA amplified product;

c3 Use as a DNA polymerase;

c4 For the preparation of DNA polymerase preparations).

5. Use of the biomaterial of claim 2 or 3 in any one of the following:

d1 Use in DNA amplification);

d2 For the preparation of a DNA amplified product;

d3 Use in the preparation of DNA polymerase;

d4 For the preparation of DNA polymerase preparations).

6. Use according to claim 4 or 5, characterized in that: the DNA amplification is DNA rolling circle amplification.

7. A reagent or kit, characterized in that: the reagent or kit comprises the protein of claim 1 or/and the biological material of claim 2 or 3.

A method for dna amplification, characterized by: the method comprising using the protein of claim 1 for DNA amplification.

9. The method according to claim 7, wherein: the template for DNA amplification is a double-stranded DNA molecule or a single-stranded DNA molecule.

10. The method according to any one of claims 7-9, characterized in that: the length of the primer in the reaction system of DNA amplification is 6-15nt.