Recombinant polymerase, encoding gene, preparation method, vector, kit and application

A polymerase and gene technology, applied in the fields of molecular biology and protein engineering, can solve the problems of TaqDNA polymerase's low continuous synthesis ability, limit the rapid application of PCR technology, and low substrate sensitivity, so as to improve the continuous synthesis ability, The effect of improving the effect of DNA amplification and the prospect of wide application

Pending Publication Date: 2021-04-16
HUBEI UNIV
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Problems solved by technology

[0007] (1) PCR technology also faces many limitations. Blood and tissue samples cannot be directly used in PCR reactions, and DNA needs to be further extracted, which limits the rapid application of PCR technology.
[0008] (2) The continuous synthesis ability of natural Taq DNA polymerase is not high, and the number of polymerization reactions catalyzed by the polymerase combined with the template is small
[0009] The difficulty in solving the above problems and defects is that the existing Taq DNA polymerases have low sensitivity to substrates, and the corresponding mutants with high sensitivity have not been found yet, and most Taq DNA polymerases currently on the market are less than Targets for direct detection of body fluids or samples

Method used

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  • Recombinant polymerase, encoding gene, preparation method, vector, kit and application
  • Recombinant polymerase, encoding gene, preparation method, vector, kit and application
  • Recombinant polymerase, encoding gene, preparation method, vector, kit and application

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preparation example Construction

[0050] Such as Figure 7 As shown, the preparation method of Cl7-Taq recombinant DNA polymerase provided by the embodiments of the present invention comprises the following steps:

[0051] S101, design and synthesize primers according to the Taq gene sequence and Cl7 gene sequence information published by GenBank, and amplify the Cl7-Taq sequence with purification tags and restriction sites by Overlap PCR technology;

[0052] S102, cloning the Cl7-Taq obtained in S101 into the expression vector pET30a to construct the recombinant vector pET30a-Cl7-Taq;

[0053] S103, the expression vector pET30a-Cl7-Taq was transformed into Escherichia coli and induced to express the target protein Cl7-Taq.

Embodiment 1

[0060] This embodiment is used to illustrate the construction of recombinant plasmid pET30a-Cl7-Taq (see figure 1 ).

[0061] The Taq gene sequence (SEQ ID NO.1) was synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., and 6 histidines were attached to its C-terminus as a tag. The target gene was amplified by PCR with primers Taq-Linker and Taq-his. The pET30a vector containing T7 promoter, ribose binding site, T7 terminator and carat resistance is used as a high-copy cloning vector of the target gene, and the Taq gene is cloned into the plasmid pET30a recombinant vector pET30a-Taq by homologous recombination; recombination The pET30a-Taq gene was transformed into competent cells and verified by DNA sequencing.

[0062] Synthesize its gene sequence according to the published Cl7 amino acid sequence, use flexible linker (SEQ ID NO.2) to connect Cl7 (SEQ ID NO.1) to the N-terminal of Taq according to the method of enzyme-free cloning, use primer Cl7-F (SEQ ID NO.2) IDNo.14...

Embodiment 2

[0065] Embodiment 2: Expression and purification of Cl7-Taq protein

[0066] This example illustrates the induced expression and purification of recombinant proteins.

[0067] 1. Transform the positive recombinant expression plasmid pET30a-Cl7-Taq verified by sequencing into Escherichia coli expression strain BL21(DE3), pick a single colony and culture it overnight in LB liquid medium containing kanamycin at 37°C .

[0068] 2. Inoculate the overnight bacterial solution into 500ml of LB liquid medium containing kanamycin at a ratio of 1 / 100, culture it on a shaker at 37°C until the OD600 value reaches 0.6, and then add IPTG to a final concentration of 0.5mmol / L. induced. At the same time, the expression vector control group was set, and the bacterial liquid was collected after induction of expression at 18°C ​​for 12 hours, centrifuged at 12,000 r / min for 10 minutes, and the supernatant was removed.

[0069] 3. Resuspend bacterial pellet in Ni-NTA lysis buffer (20mM Tris-HCl...

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Abstract

The invention belongs to the technical field of molecular biology and protein engineering, and discloses a recombinant polymerase, an encoding gene, a preparation method, a carrier, a kit and application; wherein the recombinant polymerase is derived from a hybrid DNA polymerase obtained by covalently connecting a protein Cl7 to the N end of a Taq DNA polymerase protein sequence in a Thermous aqueatus YT1 strain through a flexible connection sequence, and the amino acid sequence is shown as SEQ ID No.4; or the sequence is an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function. The expression vector pET30a-Cl7-Taq disclosed by the invention is used for converting escherichia coli and inducing expression to obtain a target protein Cl7-Taq. The continuous synthesis capability of TaqDNA polymerase in PCR nucleic acid amplification is greatly improved, and the TaqDNA polymerase has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and protein engineering, and in particular relates to a recombinant polymerase, coding gene, preparation method, carrier, kit and application. Background technique [0002] At present, with the rapid development of large-scale sequencing and gene diagnosis, the most widely used nucleic acid amplification technology is polymerase chain reaction (Polymerase Chain Reaction, PCR). PCR technology is widely used in various fields of life sciences such as molecular cloning, genome sequencing, and clinical diagnosis, which promotes the development of precision medicine and accelerates the research on the correlation between genes and genetic diseases. Nucleic acid amplification is a very important technology in biological research, medical testing, forensic identification, agriculture and other related fields. PCR technology uses cyclical changes in reaction temperature to achieve denaturation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N15/62C12P19/34
Inventor 王亚平马立新王飞倪静
Owner HUBEI UNIV
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