阅读说明：本技术 一种耐受性提高的突变型Taq DNA聚合酶及其制备方法和应用 (Mutant Taq DNA polymerase with improved tolerance as well as preparation method and application thereof ) 是由 贡怡 冯速 徐晓昱 刘来花 曹林 张力军 聂俊伟 瞿志鹏 于 2019-10-08 设计创作，主要内容包括：本发明公开一种耐受性提高的突变型Taq DNA聚合酶及其制备方法和应用,本发明所述的一种突变型Taq DNA聚合酶,其是对如SEQ ID NO.1所示的Taq DNA聚合酶的氨基酸序列中插入、取代、或缺失1个或多个氨基酸,且与SEQ ID NO.1所示的Taq DNA聚合酶相比,其杂质耐受性显著增强的氨基酸序列。本发明的重组型Taq DNA聚合酶突变体对血液、荧光染料及高离子强度的耐受性明显增强,能够直接进行血液样品的PCR反应检测,节约时间,避免假阴性。(The invention discloses mutant Taq DNA polymerase with improved tolerance as well as a preparation method and application thereof, and the mutant Taq DNA polymerase is an amino acid sequence which inserts, substitutes or deletes 1 or more amino acids in an amino acid sequence of the Taq DNA polymerase shown in SEQ ID NO.1 and has obviously enhanced impurity tolerance compared with the Taq DNA polymerase shown in SEQ ID NO. 1. The recombinant Taq DNA polymerase mutant provided by the invention has obviously enhanced tolerance to blood, fluorescent dye and high ionic strength, can be used for directly carrying out PCR reaction detection on a blood sample, saves time and avoids false negative.)

1. A mutant Taq DNA polymerase Taq-Mut, wherein the mutant has amino acid substitutions at one or more of the following amino acid positions in the sequence shown in SEQ ID NO.1, each substitution being represented by a triplet: letter-number-letter, wherein the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number indicates the amino acid used to replace the amino acid before the number: R266F, a293N, a414F, E466Q, E507K, D732E.

2. The mutant Taq DNA polymerase Taq-Mut according to claim 1, wherein the amino acid sequence is represented by SEQ ID No.3 or an amino acid sequence having 80% identity to the sequence represented by SEQ ID No.3 and having Taq DNA polymerase activity; preferably 85% identity, more preferably 90% identity, most preferably 95% identity, even more preferably 99% identity.

3. A nucleotide sequence encoding the mutant Taq DNA polymerase Taq-Mut according to claim 1.

4. The nucleotide sequence of Taq-Mut encoding the mutant Taq DNA polymerase according to claim 1, as shown in SEQ ID No. 4.

5. A recombinant vector comprising the nucleotide sequence of claim 3 or 4.

6. A recombinant cell comprising the nucleotide sequence of claim 3 or 4 or the recombinant vector of claim 5.

7. The recombinant cell of claim 6, wherein the host cell used in the recombinant cell is BL 21.

8. Use of the mutant Taq DNA polymerase Taq-Mut according to claim 1 or 2, the nucleotide sequence according to claim 3 or 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6 or 7 in the field of biotechnology.

9. Use of the mutant Taq DNA polymerase Taq-Mut according to claim 1 or 2, the nucleotide sequence according to claim 3 or 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6 or 7 in the field of PCR.

10. Use of the mutant Taq DNA polymerase Taq-Mut according to claim 1 or 2, the nucleotide sequence according to claim 3 or 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6 or 7 in the field of PCR of samples comprising blood, samples comprising SYBR Green or/and salt-containing samples.

Technical Field

The invention relates to the technical field of biology, in particular to mutant Taq DNA polymerase with improved tolerance and a preparation method and application thereof.

Background

DNA polymerases are a class of enzymes that use single-stranded DNA as a template to synthesize a complementary DNA strand. The DNA polymerase can add free nucleotides to the 3' end of the newly formed strand, extending the new strand in the 5' -3' direction. Most DNA polymerases are multifunctional proteins with polymerization and exonucleolytic activity (e.g., 3'-5' exonuclease or 5'-3' exonuclease activity).

Like other natural enzymes, DNA polymerases have been developed for millions of years to function in their natural cellular environment. Many of which are almost fully adapted to work in this environment. However, when a DNA polymerase is extracted from its natural environment and used for industrial or research, the operating environment and conditions of the enzyme inevitably differ greatly from those of its evolution, and the limiting factors of the evolution of the natural environment are largely eliminated, and therefore, improvement of the DNA polymerase for industrial or research applications has a great potential. Taq DNA polymerase, the first enzyme to be found that can be used in PCR reactions, has been very intensively studied for modification and has many biochemical and structural data available. Some of the mutation sites are critical for the fidelity of the enzyme (US6395524, US6602695 and US5614365), some of the mutations can increase its A addition efficiency (CN201611196755.9), make the enzyme less temperature sensitive (Barnes and Kermekchiev, 2000), or can increase the resistance of the polymerase to different PCR inhibitors (Kermekchiev MB, Tzekov A and Barnes WM. Cold-sensitive mutants of Taq polymerase a hot start for PCR. nucleic Acids Research,2003,31: 6139-.

This Taq DNA polymerase mutant is useful for detection of pathogens or specific substances in clinical and environmental samples, which usually contain PCR inhibitors (dyes, blood, soil), which is the main reason for the need for sample handling. However, the sample purification process is very inefficient and labor intensive, and may introduce new impurities or cause errors. The direct PCR or quantitative PCR detection is a simpler and more efficient scheme without sample treatment, but the TaqDNA polymerase is required to be capable of tolerating inhibitor components involved in the detection process, such as blood, fluorescent dye, high ionic strength and the like, and efficiently obtaining a single and accurate target strip. For example, SYBR Green intercalating dyes are used for qPCR, which will inhibit Taq DNA polymerase and reduce PCR efficiency and sensitivity. Increased resistance to SYBR Green dyes may be associated with increased resistance to enzymes from blood and other PCR inhibitors (Kermekchiev MB, Kirilova LI, Valil EE, Barnes WM. variants of Taq DNApolymerase resistance to PCR inhibition from microbial and crop soil samples, nucleic Acids Research 2009,37: e 40.).

Currently, PCR represents one of the fastest growing segments of the molecular biology application market. New applications of PCR and new variants of PCR are being developed and introduced for research and diagnostic applications such as rapid qPCR, digital PCR and direct sample-to-PCR requiring new enzyme properties. Therefore, there is a need in the industry for new and improved Taq DNA polymerase derivatives.

Blood has a significant inhibitory effect on PCR, and various DNA extraction methods have been developed to reduce the inhibitory effect of blood components on PCR, and these pretreatment steps are often time consuming, labor intensive, and adaptable only to specific samples, and are not general methods. Furthermore, even after the use of the DNA extraction kit, some PCR inhibitors may still be present. For example, human hepatitis B virus testing is performed using a blood DNA purification kit, of which about 14% may be false negative (KramvisA, Bukovzer S.and KewM.C. Complex of hepatitis B viruses DNAextractions from serum by the QIAampblod kit, Generelease, and the phenol-chlorine method J.Clin.Microbiol.,1996,34, 2731-. SYBR Green fluorescent dye is a commonly used dye for qPCR, but Taq DNA polymerase is very resistant to it.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a mutant Taq DNA polymerase with improved tolerance as well as a preparation method and application thereof.

The invention provides a mutant Taq DNA polymerase, which is an amino acid sequence which inserts, substitutes or deletes 1 or more amino acids in the amino acid sequence of the Taq DNA polymerase shown in SEQ ID No.1 and has obviously enhanced impurity tolerance compared with the Taq DNA polymerase shown in SEQ ID No. 1.

In one embodiment of the invention, the mutant Taq DNA polymerase (denoted by Taq-Mut) according to the invention has amino acid substitutions at one or more of the following amino acid positions in the sequence shown in SEQ ID No.1, each substitution being denoted by a triplet: letter-number-letter, wherein the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number indicates the amino acid used to replace the amino acid before the number: R266F, a293N, a414F, E466Q, E507K, D732E.

R266F represents the change of the amino acid 226 from arginine to phenylalanine; A293N shows that amino acid 293 was changed from alanine to asparagine; a414F indicating that amino acid 414 was changed from alanine to phenylalanine; E466Q shows that the 466 th amino acid has been changed from glutamic acid to glutamine; E507K shows that amino acid 507 is changed from glutamic acid to lysine; D732E shows that amino acid 732 was changed from aspartic acid to glutamic acid.

In one embodiment of the invention, the amino acid sequence of the mutant Taq DNA polymerase Taq-Mut is shown in SEQ ID NO.3, or the amino acid sequence with Taq DNA polymerase activity and having 80% identity with the sequence shown in SEQ ID NO. 3; preferably 85% identity, more preferably 90% identity, most preferably 95% identity, even more preferably 99% identity.

In another aspect, the invention provides methods for engineering modifications to Taq DNA polymerase based on the various mutations described herein.

In one embodiment of the invention, the invention also provides a nucleotide sequence encoding the mutant Taq DNA polymerase Taq-Mut according to the invention. It should be noted that, since the same amino acid may be determined by a plurality of different codons, the nucleotide sequence encoding the variant Taq DNA polymerase Taq-Mut may be a nucleotide sequence encoding the same amino acid sequence as that in SEQ ID NO.3 obtained by mutating one or more nucleotides from the nucleotide sequence of the wild type Taq DNA polymerase shown in SEQ ID NO.2 to form a synonymous mutation. In a specific embodiment, the nucleotide sequence encoding the mutant Taq DNA polymerase Taq-Mut of the present invention is shown in SEQ ID NO. 4.

The invention also provides a recombinant vector containing a nucleotide sequence for encoding the mutant Taq DNA polymerase Taq-Mut. In a specific embodiment, the recombinant vector comprises the nucleotide sequence shown as SEQ ID No. 4.

The invention also comprises a recombinant cell which comprises a nucleotide sequence which can code the mutant Taq DNA polymerase Taq-Mut or a vector which comprises a nucleotide sequence which can code the mutant Taq DNA polymerase Taq-Mut.

Further, the host cell used for the recombinant cell is BL 21.

The invention also provides a preparation method of the mutant Taq DNA polymerase Taq-Mut capable of improving the tolerance of impurities, which comprises the following steps:

1) constructing a vector containing a nucleotide sequence encoding a mutant Taq enzyme: carrying out PCR amplification on plasmids containing wild Taq DNA polymerase genes by using primers containing mutation site information to obtain target bands, then obtaining sufficient plasmids by using homologous recombination or directly converting to obtain mutant plasmids, and sequencing to verify that the nucleotide sequences are correct;

2) transforming the vector obtained in the step 1) into a host cell to obtain a recombinant cell; transforming the vector containing the correct nucleotide sequence into a host cell, and screening by using antibiotics to obtain a correct recombinant cell;

3) culturing, expressing and collecting recombinant cells, and extracting and purifying mutant Taq enzyme.

Further, in step 1), the specific sequences of the primers are as follows:

R266F-1：CGGGAGCCCGACTTCGAGAGGCTTAGGGCCTTTCTG(SEQ ID NO.5)；

R266F-2：CCTAAGCCTCTCGAAGTCGGGCTCCCGCCTTTTGGC(SEQ ID NO.6)；

A293N-1：GAAAGCCCCAAGAACCTGGAGGAGGCCCCCTGGCCC(SEQ ID NO.7)；

A293N-2：GGCCTCCTCCAGGTTCTTGGGGCTTTCCAGAAGGCC(SEQ ID NO.8)；

A414F-1：GAGAGGCTCTTCTTCAACCTGTGGGGGAGGCTTGAG(SEQ ID NO.9)；

A414F-2：CCCCCACAGGTTGAAGAAGAGCCTCTCGGAAAGGGC(SEQ ID NO.10)；

E466Q-1：GAGGTGGCCGAGCAGATCGCCCGCCTCGAGGCCGAG(SEQ ID NO.11)；

E466Q-2：GAGGCGGGCGATCTGCTCGGCCACCTCCAGGGACAA(SEQ ID NO.12)；

E507K-1：ATCGGCAAGACGAAGAAGACCGGCAAGCGCTCCACC(SEQ ID NO.13)；

E507K-2：CTTGCCGGTCTTCTTCGTCTTGCCGATGGCGGGAAG(SEQ ID NO.14)；

D732E-1：CGCTACGTGCCAGAGCTAGAGGCCCGGGTGAAGAGC(SEQ ID NO.15)；

D732E-2：CCGGGCCTCTAGCTCTGGCACGTAGCGGCGGCGGCC(SEQ ID NO.16)。

the above primers of the present invention contain the desired mutation site and the nucleotide after substitution.

The invention also provides application of the mutant Taq DNA polymerase Taq-Mut in the field of biotechnology. More particularly preferred is the use in the field of PCR. In particular, in some embodiments, the invention relates to the use of mutant Taq DNA polymerase Taq-Mut in the field of PCR of samples comprising blood, samples comprising SYBR Green, or/and saline samples.

The invention has the beneficial effects that:

(1) the invention carries out directional modification for improving impurity tolerance on the existing wild type Taq DNA polymerase to construct mutant Taq DNA polymerase, which can tolerate higher blood sample concentration, SYBR Green concentration and salt ion concentration, and shows higher polymerization amplification capability and lower false negative rate. The impurity tolerance of the mutant Taq DNA polymerase is improved.

(2) The mutant Taq DNA polymerase can be amplified in a PCR system containing 20% of blood, the product amplification has no obvious difference from the blood-free condition, the purification step of blood sample detection is omitted, the mutant Taq DNA polymerase can be applied to the rapid amplification of a large number of unpurified samples, and the dosage of the required enzyme is reduced by at least 3 times compared with that of the wild Taq DNA polymerase.

(3) Mutant Taq DNA polymerases are at least 4-fold more tolerant to SYBR Green dye than wild-type Taq DNA polymerases, mutants that are capable of faster and/or more efficient DNA amplification compared to wild-type enzymes.

(4) The tolerance of the mutant Taq DNA polymerase to the KCl concentration in a PCR system is at least 3 times higher than that of the wild Taq DNA polymerase, and the amplification is not influenced in an amplification system containing 150mM KCl.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection of the amplification product of example 3 of the present invention. Wherein, lanes 1-3 are amplification results of Taq DNA polymerase, and the enzyme adding amount is respectively 60ng, 40ng and 20ng in sequence; lanes 4-6 are the amplification results of mutant Taq DNA polymerase, in which the amounts of enzyme added are 60ng, 40ng and 20ng, respectively;

FIG. 2 shows the result of agarose gel electrophoresis detection of the amplification product of example 4 of the present invention. Wherein, lanes 1-2 are the amplification result of Taq DNA polymerase, and the final concentration of KCl is respectively 50mM and 150mM in sequence; lanes 3-4 are the amplification results of mutant Taq DNA polymerase, wherein the final KCl concentrations are 50mM and 150mM respectively in sequence;

FIG. 3 shows the result of agarose gel electrophoresis detection of the amplification product of example 5 of the present invention. Wherein lanes 1-5 are amplification results of Taq DNA polymerase, wherein the final concentrations of SYBR Green dyes are 0X, 1X, 3X, 4X and 5X respectively in sequence; lanes 6-10 are the amplification results of mutant Taq DNA polymerase, where the final concentrations of SYBR Green dye were 0 ×,1 ×,3 ×, 4 ×, and 5 ×, respectively.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental procedures without specific conditions being noted, generally according to means well known in the art, such as molecular cloning, which is described in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.