阅读说明：本技术 Dna聚合酶及其制备方法、表达基因、表达载体、宿主细胞及试剂盒 (DNA polymerase, preparation method thereof, expression gene, expression vector, host cell and kit ) 是由 高亚平 田晖 何筠 于 2019-08-22 设计创作，主要内容包括：本发明涉及一种DNA聚合酶及其制备方法、表达基因、表达载体、宿主细胞及试剂盒。该DNA聚合酶包括氨基酸序列如SEQ ID No.1所示的蛋白片段,该DNA聚合酶具有较高的耐盐性。(The invention relates to a DNA polymerase, a preparation method thereof, an expression gene, an expression vector, a host cell and a kit. The DNA polymerase comprises a protein fragment with an amino acid sequence shown as SEQ ID No.1, and has high salt tolerance.)

1. An expression gene of DNA polymerase, which is characterized by comprising a nucleotide fragment of a protein fragment with an amino acid sequence shown as SEQ ID No. 1.

2. The gene expressed by the DNA polymerase of claim 1, comprising a nucleotide fragment having the sequence shown in SEQ ID No. 2.

3. An expression vector for a DNA polymerase, comprising the expression gene for a DNA polymerase according to claim 1 or 2.

4. A host cell comprising the DNA polymerase expression vector of claim 3.

5. A DNA polymerase comprising a protein fragment having an amino acid sequence as set forth in SEQ ID No. 1.

6. A method for preparing a DNA polymerase, comprising the steps of:

Constructing an expression vector for expressing the DNA polymerase by using an expression gene of the DNA polymerase and an empty vector, wherein the expression gene of the DNA polymerase comprises a nucleotide fragment of a protein fragment with an amino acid sequence shown as SEQ ID No. 1; and

And transferring the expression vector into a host cell, and performing induction culture to obtain the DNA polymerase.

7. The method of claim 6, wherein the empty vector is pGEX-6P-1; and/or

The host cell is a prokaryotic cell.

8. A DNA amplification kit comprising the DNA polymerase according to claim 5 or the DNA polymerase produced by the method for producing a DNA polymerase according to any one of claims 6 to 7.

9. the DNA amplification kit according to claim 8, further comprising at least one of a nucleic acid extraction reagent and a PCR buffer.

10. A nanopore sequencing kit, comprising a sequencing component having a nanopore, and a detection reagent, wherein the detection reagent comprises the DNA polymerase according to claim 5 or the DNA polymerase prepared by the method for preparing the DNA polymerase according to any one of claims 6 to 7.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a DNA polymerase, a preparation method thereof, an expression gene, an expression vector, a host cell and a kit.

Background

The discovery of DNA polymerases is an important milestone in recent biotechnology. The DNA polymerase can complete the replication of nucleic acid chain by using DNA as template and dNTPs as substrate. In recent years, DNA polymerases have been widely used in various fields of biotechnology and scientific research, including cDNA synthesis, DNA end modification, and DNA sequencing, and more thermostable DNA polymerases and isothermal DNA polymerases have been developed and used.

However, the salt tolerance of the current DNA polymerases is relatively poor. A general DNA polymerase can tolerate sodium ions below 200mM, and when the concentration of the sodium ions exceeds 200mM, the activity of the DNA polymerase is sharply reduced or even completely lost.

disclosure of Invention

Therefore, it is necessary to provide a DNA polymerase with better salt tolerance and a preparation method thereof, aiming at the problem of poor salt tolerance of the traditional DNA polymerase. In addition, it is necessary to provide an expression gene, an expression vector, a host cell and a kit for expressing the DNA polymerase.

An expression gene of DNA polymerase, which comprises a nucleotide fragment of a protein fragment with an amino acid sequence shown as SEQ ID No. 1.

The expression gene of the DNA polymerase comprises a nucleotide fragment of which the coding amino acid sequence comprises a protein fragment shown as SEQ ID No.1, and the DNA polymerase expressed by the expression gene of the DNA polymerase has good salt tolerance.

In one embodiment, the nucleotide fragment comprises a nucleotide fragment with a sequence shown as SEQ ID No. 2.

An expression vector for a DNA polymerase, comprising an expression gene for the above DNA polymerase.

A host cell comprising the expression vector for the DNA polymerase.

A DNA polymerase comprises a protein fragment with an amino acid sequence shown as SEQ ID No. 1.

a method for preparing a DNA polymerase, comprising the steps of:

constructing an expression vector for expressing the DNA polymerase by using an expression gene of the DNA polymerase and an empty vector, wherein the expression gene of the DNA polymerase comprises a nucleotide fragment of a protein fragment with an amino acid sequence shown as SEQ ID No. 1; and

And transferring the expression vector into a host cell, and performing induction culture to obtain the DNA polymerase.

In one embodiment, the empty vector is pGEX-6P-1; and/or

The host cell is a prokaryotic cell.

A DNA amplification kit comprising the above DNA polymerase or the DNA polymerase prepared by the above DNA polymerase preparation method.

In one embodiment, the kit further comprises at least one of a nucleic acid extraction reagent and a PCR buffer.

A nanopore sequencing kit comprises a sequencing component with a nanopore and a detection reagent, wherein the detection reagent comprises the DNA polymerase or the DNA polymerase prepared by the preparation method of the DNA polymerase.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of the DNA polymerase of example 1;

FIG. 2 is an SDS-PAGE electrophoresis of Phi29DNA polymerase of example 2;

FIG. 3 is a graph showing the effect of temperature on the activity of the DNA polymerase prepared in example 1;

FIG. 4 is a graph showing the effect of metal ions on the activity of the DNA polymerase prepared in example 1;

FIG. 5 is a graph showing the results of buffer1, buffer1 supplemented with 300mM NaCl, and a blank control;

FIG. 6 is a graph showing the results of buffer2, buffer2 supplemented with 300mM NaCl, and a blank control;

FIG. 7 is a graph showing the results of buffer3, buffer3 supplemented with 300mM NaCl, and a blank control;

FIG. 8 is a graph showing the results of buffer4, buffer4 supplemented with 300mM NaCl, and a blank control;

FIG. 9 is a graph showing the results of salt tolerance of the DNA polymerase prepared in example 1;

FIG. 10 is a graph showing the results of salt tolerance of the Phi29DNA polymerase prepared in example 2;

FIG. 11 is a graph showing the results of salt tolerance of NEB Taq DNA polymerase;

FIG. 12 is a graph showing the quantitative measurement of the enzyme activities of the DNA polymerase prepared in example 1, the Phi29DNA polymerase prepared in example 2, and NEB Taq DNA polymerase;

FIG. 13 is a graph showing the continued comparison of Phi29DNA polymerase with the DNA polymerase prepared in example 1.

Detailed Description

to facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the present invention provides an expression gene of a DNA polymerase, wherein the expression gene of the DNA polymerase includes a nucleotide fragment encoding a protein fragment having an amino acid sequence represented by SEQ ID No.1, and the DNA polymerase expressed by the expression gene of the DNA polymerase has good salt tolerance.

Specifically, the expression gene of the DNA polymerase comprises a nucleotide fragment of a protein fragment with an amino acid sequence shown as SEQ ID No. 1. The amino acid sequence shown as SEQ ID No.1 is as follows:

MSELKDYSEYLPKFLLDLDPEIYLSMNFLVLDLETDTDGDERSPDATWEQNDIVSASWVFGTGGRGTEKFVYGGIHDMDELIQDMYEADFVVAHNAKFDIKWLIRAGLDPSRILVADTMLAEYVLTGNLKAGKKGALTLGALAKQYLGVTKDPLVDKLMRGGVSPRVIPKSLLERRNKSDIFQTRNLWLKLRDEMMERDVIHLFYNRCLLSPVLADIELNGVHLDKERVCEEHDIASRRMAEVEADLMDMIDGRNPRSVPQMQEFIYDVLKFKPLKKRGVEWRPTGEEVLQFEARTKKQQAFLDLKKEFAQLNADLSKNLDYFYGVVTEREDCLFYAQFNQAQTVTHRLSSSGIKTKFEMYPKAKSIQLQNSPRKYKRLYSARNPDWYVIEMDGAQIEFRVAGYIGQDTRICQDIVDGVDVHRFTASVLNHCDEEEVTKDQRTDAKPDTFKPLYGGQYGTDDQMAYYEAFRNKYQDITQAQQDWLMKVLRNKEITHPTGITFYYPNASMSSSGYCQDFPSVCNYPVQNLATAEIIPIALVAIWHVMKAMKLQSFLVNTVHDSVISESPRDELELMYEISKWAFLWWVYEFLDICYDLQFNVPLGVGYQAHTHWGGGKEIFFEPSQYDGEVVKIDKGEITVTAIPPTKMDGVDYSELYKGDK。

In one embodiment, the expression gene of the DNA polymerase is a nucleotide fragment encoding a protein fragment having an amino acid sequence shown in SEQ ID No. 1.

Further, the expression gene of the DNA polymerase comprises a nucleotide fragment with a sequence shown as SEQ ID No. 2. Specifically, the nucleotide sequence shown as SEQ ID No.2 is:

5’-ATGAGTGAGCTGAAAGACTACAGCGAGTACCTACCTAAGTTCCTATTGGACTTAGACCCTGAGATATACCTGTCCATGAACTTCCTTGTCCTTGACTTGGAGACAGACACGGATGGGGATGAGCGTTCGCCTGATGCTACATGGGAACAGAACGATATTGTTTCTGCCTCATGGGTGTTCGGTACAGGTGGACGTGGTACTGAGAAGTTTGTCTATGGTGGTATCCATGACATGGACGAACTGATACAGGACATGTACGAAGCAGACTTTGTCGTGGCACACAATGCTAAGTTCGACATCAAGTGGCTCATCCGTGCAGGGCTAGACCCTTCACGTATCCTAGTAGCTGACACGATGCTGGCTGAGTATGTGCTTACGGGCAATCTCAAAGCAGGTAAGAAGGGTGCGCTTACGCTAGGTGCTCTGGCTAAGCAATACCTCGGTGTTACTAAAGACCCCCTCGTTGATAAGTTGATGCGAGGTGGGGTGTCACCACGTGTGATACCTAAGTCTTTACTTGAACGCCGTAACAAATCAGACATCTTCCAGACAAGAAACCTGTGGCTCAAGCTACGTGATGAGATGATGGAACGTGATGTGATACATCTGTTCTATAACCGTTGCTTGCTGTCCCCCGTGTTAGCCGACATTGAGTTGAACGGGGTGCATCTGGATAAGGAACGAGTATGTGAGGAACATGACATAGCCTCTCGCCGTATGGCTGAGGTGGAAGCTGACTTGATGGACATGATAGACGGACGTAACCCCCGCTCTGTACCGCAGATGCAGGAGTTCATCTACGATGTGCTCAAGTTCAAGCCATTAAAGAAACGTGGTGTTGAGTGGCGACCAACAGGGGAGGAGGTACTGCAATTCGAGGCACGTACCAAGAAGCAGCAGGCTTTCCTTGACCTCAAGAAAGAGTTCGCTCAGCTCAATGCTGACCTAAGTAAGAACCTCGACTACTTCTACGGGGTAGTCACGGAACGTGAGGACTGTTTATTCTATGCACAATTCAACCAAGCTCAGACCGTTACGCACAGGTTGTCTTCTTCGGGTATTAAGACCAAGTTCGAAATGTACCCGAAAGCCAAGAGCATTCAGTTACAGAACTCACCTCGAAAATATAAGCGTCTTTACTCAGCCCGAAATCCCGACTGGTACGTCATTGAAATGGACGGTGCTCAGATTGAATTTAGGGTTGCTGGATATATAGGACAAGACACGCGCATCTGTCAGGATATTGTTGACGGTGTAGACGTGCACCGATTCACCGCCTCAGTGTTGAACCACTGTGACGAGGAGGAGGTAACTAAGGACCAGCGTACTGACGCTAAGCCTGATACATTCAAGCCACTGTATGGAGGACAGTATGGTACGGATGACCAGATGGCTTACTACGAAGCGTTCCGTAACAAGTATCAAGACATCACACAGGCACAACAAGACTGGCTGATGAAGGTGCTACGTAACAAAGAGATTACCCATCCAACGGGTATCACTTTCTACTACCCTAACGCCAGCATGAGTAGCAGTGGTTACTGTCAGGACTTCCCGTCCGTGTGTAACTACCCAGTGCAGAACCTAGCGACAGCGGAAATCATCCCTATCGCATTGGTTGCTATCTGGCATGTGATGAAAGCAATGAAGCTACAGTCGTTCTTAGTTAACACTGTGCATGACTCAGTAATATCTGAATCACCACGTGATGAGTTGGAGCTGATGTACGAGATTAGTAAGTGGGCTTTCCTCTGGTGGGTCTACGAATTTCTGGACATCTGCTATGATTTACAATTCAATGTCCCGCTCGGTGTTGGTTATCAGGCTCACACCCATTGGGGTGGTGGTAAGGAAATCTTCTTCGAGCCTTCGCAGTATGACGGCGAGGTTGTGAAGATAGACAAAGGTGAGATAACTGTTACAGCTATCCCGCCTACTAAGATGGATGGAGTTGATTACTCCGAACTGTATAAGGGAGACAAGTAA-3’。

Of course, in other embodiments, the nucleotide segment encoding the protein segment with the amino acid sequence shown in SEQ ID No.1 is not limited to the nucleotide segment with the sequence shown in SEQ ID No.2, but can be other nucleotide segments capable of encoding the protein segment with the amino acid sequence shown in SEQ ID No.1 according to the degeneracy of codons.

An embodiment of the present invention also provides an expression vector of a DNA polymerase for expressing the expression gene of the DNA polymerase. The DNA polymerase obtained by expression of the expression vector of the DNA polymerase has good salt tolerance.

In one embodiment, the expression vector of the DNA polymerase includes an empty vector and an expression gene of the DNA polymerase inserted into the empty vector. The empty vector has an expression element and a multiple cloning site for facilitating insertion of the expressed gene of the above-mentioned DNA polymerase. The expression element is used for expressing the expression gene of the DNA polymerase. Further, the empty vector may further comprise a purification tag, such as a histidine tag, a GST tag, etc. The purification tag is used for purifying the DNA polymerase expressed by the expression gene of the above DNA polymerase. Further, the empty vector is pGEX-6P-1. pGEX-6P-1 has a GST tag, facilitating subsequent purification of DNA polymerase.

An embodiment of the present invention also provides a host cell comprising the above-described expression vector for a DNA polymerase. The host cell can produce DNA polymerase which has good salt tolerance and is suitable for amplification reaction with high salt ion concentration.

In one embodiment, the host cell is a prokaryotic cell. Further, the host cell is Escherichia coli.

The invention also provides a DNA polymerase, which comprises a protein fragment with an amino acid sequence shown as SEQ ID No. 1. The DNA polymerase has good salt tolerance and is suitable for amplification reaction with high salt ion concentration. In addition, the DNA polymerase enables an amplification reaction to be performed under a relatively low temperature condition. Of course, it is also suitable for preparing nanopore sequencing kits.

an embodiment of the present invention further provides a method for preparing a DNA polymerase, including steps S110 to S130.

Step S110, constructing an expression vector for expressing the DNA polymerase by using the expression gene of the DNA polymerase and the empty vector, wherein the expression gene of the DNA polymerase comprises a nucleotide fragment of a protein fragment with an amino acid sequence shown as SEQ ID No. 1.

specifically, step S110 includes steps S111 to S113.

Step S111, providing the expression gene of DNA polymerase. The expression gene of the DNA polymerase comprises a nucleotide fragment and a restriction enzyme site of a protein fragment with an encoded amino acid sequence shown as SEQ ID No. 1.

Step S113, constructing an expression vector containing the expression gene of the DNA polymerase.

Specifically, the expression vector containing the expression gene of the DNA polymerase is obtained by introducing the expression gene of the DNA polymerase into an empty vector.

Further, the expression gene of the DNA polymerase is digested and ligated to an empty vector after the digestion treatment, thereby obtaining an expression vector containing the expression gene of the DNA polymerase. The digestion treatment can adopt NotI/EcoRI double digestion. The empty vector can be selected from various conventional standard expression vectors according to the expression sequence characteristics of the expression gene of the DNA polymerase and conventional molecular cloning experimental guidelines.

In one embodiment, the empty vector is pGEX-6P-1.

And S130, transferring the expression vector into a host cell, and performing induction culture to obtain the DNA polymerase.

Specifically, step S130 includes steps S131 to S137.

And S131, transforming the expression vector into a host cell to obtain a recombinant cell.

Specifically, the expression vector transformation can be achieved by methods recommended by the kit manufacturer.

The host cell is a competent cell, e.g., an E.coli competent cell. In this embodiment, the host cell is an escherichia coli competent cell, Rossete.

In one embodiment, the constructed gene expression vector is added into a competent cell, heat shock treatment is carried out to disturb the cell membrane structure of the competent cell, and gaps appear on the cell membrane so that the gene expression vector can enter the cell, and then constant temperature culture is carried out to recover the host cell.

further, after the expression vector is transformed into the host cell, the method also comprises a step of performing resistance screening on the transformed host cell. Specifically, the type of antibiotic added to the medium may be selected according to the resistance gene on the expression vector.

Step S133, performing inducible expression on the recombinant cells obtained in step 131, and collecting cells.

specifically, recombinant cells are cultured, then an inducer is added to induce and express the recombinant cells, and then the cells are collected by centrifugation.

Further, the inducer is isopropyl thiogalactoside (IPTG). The temperature of the induced expression is 16-28 ℃, the final concentration of the inducer is 100-1000 mu M, and the time of the induced expression is 4-20 h. Furthermore, the temperature of the induced expression is 16-25 ℃, the final concentration of the inducer is 100-500 mu M, and the time of the induced expression is 16-20 h.

in this embodiment, the induction conditions are IPTG at a final concentration of 300. mu.M, incubated overnight at 16 ℃.

Step S135, the bacterial cells obtained in step S133 are lysed, and a crude lysate is collected.

Specifically, the cleavage is a DNA polymerase released from the cells. Lysozyme and a detergent can be added into the thalli, and the thalli are shaken, mixed evenly and then centrifuged to collect a cracking crude product.

In this embodiment, the bacterial cells were vortexed and mixed with a PBS solution, and then disrupted by an ultrasonic cell disruptor. Wherein the power of the ultrasonic wave is 50W-100W, the ultrasonic wave is 5 s-20 s, the interval is 5 s-20 s, and the ultrasonic wave lasts for 10 min-40 min.

and step 137, purifying the cleavage crude product obtained in the step S135 to obtain the DNA polymerase.

Specifically, the cleavage crude product obtained in step S135 is purified by affinity chromatography.

In this embodiment, the crude lysate obtained in step S135 is purified using a glutaminone Sepharose 4B affinity column.

An embodiment of the present invention also provides a DNA amplification kit comprising the above DNA polymerase. The DNA amplification kit comprises the DNA polymerase, has higher salt resistance, and is suitable for DNA amplification reaction when the salt ion concentration is higher.

In one embodiment, the DNA amplification kit further comprises at least one of a nucleic acid extraction reagent and a PCR buffer. The nucleic acid extraction reagent is used for extracting DNA in a sample; PCR buffer was used for DNA amplification reaction.

Further, the nucleic acid extraction reagent comprises lysis solution and a purification agent. The lysate is used to lyse the cells or nuclei, releasing the nucleic acids. Purification agents are used to purify nucleic acids. PCR buffer (10X) containing 200 mM-500 mM Tris-HCl, 20 mM-200 mM Mg²⁺100 mM-400 mM of (NH)₄)₂SO₄40 mM-200 mM DTT.

The invention also provides a nanopore sequencing kit, which comprises a sequencing component with a nanopore and a detection reagent, wherein the detection reagent comprises the DNA polymerase.

Nanopore sequencing is a fourth generation sequencing technology recognized worldwide. According to the nanopore sequencing technology, an electric field is applied to two ends of a nanopore, nucleic acid molecules pass through the nanopore under the driving of the electric field to generate a differential current signal, and meanwhile, DNA polymerase integrates the nucleotide molecules according to template strand sequence information and completes replication, so that the sequencing while synthesis is realized. Researches show that the concentration of a salt solution in an electrolytic cell is increased during nanopore sequencing, so that the signal to noise ratio in the testing process and the accuracy of a detection result are improved, and the sequencing quality is improved. However, the conventional DNA polymerase has poor salt tolerance, so if the concentration of the salt solution of the electrolytic bath is increased, the activity of the DNA polymerase is decreased, the synthesis rate of DNA is decreased, and the rate of the whole sequencing is decreased.

The nanopore sequencing kit comprises the DNA polymerase with better salt tolerance, and can improve the accuracy and the sequencing speed of nanopore sequencing.

Specifically, the detection reagent includes the above-mentioned DNA polymerase. Further, the detection reagent also comprises a buffer solution. The buffer solution comprises 20 mM-200 mM Tris-HCl and 2 mM-20 mM Mg²⁺10 mM-40 mM of (NH)₄)₂SO₄4 mM-20 mM DTT and 0.2 mM-0.5 mM dNTPs.

Specifically, an external voltage is applied across the nanopore, and ions in the cell pass through the nanopore, producing a pA-level current. DNA polymerase takes DNA as a template, and extends and synthesizes a new chain by using dNTPs which pass through the nanopore and carry the label, and the dNTPs carrying the label generate current changes with different degrees when passing through the nanopore, thereby completing sequence determination.

Further, the reaction system for nanopore sequencing comprises a nucleic acid template of 50 nM-500 nM, the DNA polymerase of 100 nM-500 nM, Tris-HCl of 20 mM-50 mM, Mg of 2 mM-20 mM²⁺10 mM-40 mM of (NH)₄)₂SO₄4 mM-20 mM DTT and 0.2 mM-0.5 mM dNTPs. The reaction system can improve the reaction efficiency, save the reagent dosage and reduce the cost. Further, the amplification reaction system comprises 50nM to 500nM of the nucleic acid template, 100nM to 500nM of the DNA polymerase, 20mM to 100mM of Tris-HCl, 2mM to 10mM of Mg²⁺10 mM-20 mM of (NH)₄)₂SO₄4 mM-10 mM DTT, 0.2 mM-0.5 mM dNTPs and 0 mM-500 mM NaCl or 0 mM-500 mM KCl.

further, the conditions of the amplification reaction: 30-37 ℃ for 5-120 min. Under the condition of 30-37 ℃, the DNA polymerase in the amplification system has high efficiency and high fidelity.