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A kind of recombinant reca* protein and its expression method and application

A protein and recombinant vector technology, applied in the field of genetic engineering, can solve the problems of limited amplification efficiency, weak binding ability of RecA to DNA, etc., and achieve the effect of improving the efficiency of RAA amplification reaction and increasing the efficiency.

Active Publication Date: 2021-11-23
深圳易致生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the binding ability of RecA to DNA is weak in vitro, which directly limits the amplification efficiency.

Method used

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  • A kind of recombinant reca* protein and its expression method and application
  • A kind of recombinant reca* protein and its expression method and application
  • A kind of recombinant reca* protein and its expression method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Recombination reaction with recombinant RecA* protein, the reaction conditions are as follows:

[0042] Tris-HCl (pH8.3) 50mM, KCl 60mM, dithiothreitol 2mM, betaine 1M, trehalose 5.5%, PEG5.5%, BSA 0.1mg / mL; the reaction enzyme system contains dNTPs 200μM, polymer Enzyme Bsu 50U, single-chain RecA* protein 262ng / μL, E.coli RecA protein 360ng / μL; DNA substrate (Proteus mirabilis) 50ng, the concentration of upstream primer (FP) and downstream primer (RP) are both 200nM; magnesium chloride, concentration 5mM.

[0043] Upstream primer (SEQ ID NO.4): TATTCCTAAATATAGTCCAAGTTTCTTGTGATGT

[0044] Downstream primer (SEQ ID NO. 5): ATTAAAACGAAAAATATTAAGAATATCGACCCC.

[0045] Incubation was continued for 30 min at 37°C and the reaction was extracted with phenol chloroform and precipitated with ethanol. Then the amplification results were observed on 3% agarose gel and the amplification products were sequenced. The sequence of the sequenced product is SEQ ID

[0046]

Embodiment 2

[0052] Use recombinant RecA* protein for recombination reaction, the reaction conditions are as follows: Tris-HCl (pH8.3) 50mM, KCl60mM, dithiothreitol 2mM, betaine 1M, trehalose 5.5%, PEG 5.5%, BSA 0.1mg / mL ; The reaction enzyme system includes dNTPs 200 μM, polymerase Bsu 50U, single-chain binding protein 262ng / μl, E.coli RecA protein 360ng / μl; the concentration of upstream primer (FP) and downstream primer (RP) is 200nM; magnesium chloride, concentration 5mM. Incubation was continued for 30 min at 37°C and the reaction was extracted with phenol chloroform and precipitated with ethanol. Amplification results were observed on 4% agarose gel.

[0053] Upstream primer (SEQ ID NO.6): ATTTCATAACCCCCCTGTTTATTGTGTTAATGG

[0054] Downstream primer (SEQ ID NO.7): ATTTTGTTAAACTCAAACGTGATAATGAGAAT

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Abstract

The invention discloses a recombinant RecA* protein, which is a polypeptide obtained by modifying the 62nd, 69th, 73rd and 85th amino groups in the amino acid sequence of Pseudomonas aeruginosa RecA, named recombinant RecA* protein , the polypeptide of the recombinant RecA* protein has the amino sequence of SEQ ID NO.1. Compared with the natural RecA protein, the recombinant RecA* protein of the present invention has stronger catalytic ATP hydrolysis efficiency, stronger binding ability with DNA, and can form a more stable protein-DNA complex; the recombinant RecA protein is applied to RAA In the amplification detection kit, the efficiency of the RAA amplification reaction can be greatly improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant RecA* protein sequence and its expression method and application Background technique [0002] In vitro nucleic acid rapid amplification technology is a technology that can efficiently and rapidly amplify trace amounts of nucleic acid in vitro. and wider adaptability. The recombinase-mediated amplification method is a constant temperature in vitro rapid nucleic acid amplification technology developed on the basis of the existing in vitro nucleic acid amplification principle. [0003] Recombinase nucleic acid isothermal amplification method (RAA) uses recombinase, single-strand binding protein and DNA polymerase to replace the thermal cycle melting process of traditional PCR, and realizes rapid nucleic acid amplification at a constant temperature of 37°C without special assistance The instrument does not have high requirements for operators. It is simpl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/63C12Q1/6844
CPCC12N9/00C12Q1/6844C12Q2531/119C12Q2521/507C12Q2522/101C12Q2537/1376
Inventor 李腾宋又强吴永辉李达
Owner 深圳易致生物科技有限公司
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