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A kind of drug-resistant Escherichia coli integrase activity assay method

A technology for the determination of Escherichia coli and activity, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of bacterial multi-drug resistance and other issues

Active Publication Date: 2021-07-16
河套学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The in vitro integration activity method of gene therapy tool enzyme phiC31 has been established, and the in vitro activity detection method of HIV-1 integrase has also been established, but the in vitro activity detection method of bacterial drug resistance integrase has not been reported. Bacteria capture foreign drug resistance through integration Genes make bacteria resistant and multi-drug resistant, and cause the spread of multi-drug resistance in bacteria. Therefore, the activity of integrase often reflects the strength of bacterial drug resistance. The integrase expressed by bacteria through the integron system can make The integration of the attI and attC sites changes the length of the DNA sequence between the attI and attC sites of the same chain, so the integration efficiency of the integrase reflects the activity of the integrase

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  • A kind of drug-resistant Escherichia coli integrase activity assay method
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  • A kind of drug-resistant Escherichia coli integrase activity assay method

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Embodiment 1

[0031] A method for assaying drug-resistant Escherichia coli integrase activity, comprising the following steps:

[0032] S1, extraction of Escherichia coli plasmid DNA, which contains the integron gene intI encoding integrase;

[0033] S2, using the plasmid DNA in S1 as a template, design specific primers, the primer sequence is:

[0034] Upstream primer Int-F: 5'-ccggaattccggacgcgtctgacccccaa-3', as shown in SEQ ID NO.1;

[0035] Downstream primer Int-R: 5'-cccaagcttgggcccgccgctgagatgc-3', as shown in SEQ ID NO.2;

[0036] The integrase gene intI encoding integrase was amplified by PCR and connected to the expression vector pEASY-E1 to obtain the recombinant vector pEASY-intI, and then pEASY-intI was transformed into Escherichia coli BL21 (DE3) to obtain transformed Escherichia coli BL21 (DE3), in order to verify whether the vector transformation is successful, the transformed Escherichia coli BL21 (DE3) can be induced to express integrase, and after purification, SDS-poly...

Embodiment 2

[0052] A method for assaying drug-resistant Escherichia coli integrase activity, comprising the following steps:

[0053] S1, extracting Escherichia coli type I plasmid DNA, which contains the integron gene intI encoding integrase;

[0054] S2, using the plasmid DNA in S1 as a template, design specific primers, the primer sequence is:

[0055] Upstream primer Int-F: 5'-ccggaattccggacgcgtctgacccccaa-3', as shown in SEQ ID NO.1;

[0056] Downstream primer Int-R: 5'-cccaagcttgggcccgccgctgagatgc-3', as shown in SEQ ID NO.2;

[0057] The integrase gene intI encoding integrase was amplified by PCR and connected to the expression vector pEASY-E1 to obtain the recombinant vector pEASY-intI, and then pEASY-intI was transformed into Escherichia coli BL21 (DE3) to obtain transformed Escherichia coli BL21 (DE3), in order to verify whether the vector transformation is successful, the transformed Escherichia coli BL21 (DE3) can be induced to express integrase, and after purification, SDS-...

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Abstract

The present invention relates to the technical field of measuring biological enzyme activity, in particular to a method for measuring drug-resistant Escherichia coli integrase activity, comprising: extracting Escherichia coli plasmid DNA; using the plasmid DNA as a template, PCR amplifies an integrase gene encoding integrase intI, and connected to the expression vector pEASY‑E1, and then transformed into Escherichia coli BL21 (DE3); PCR amplified the DNA fragment containing the integrase gene recombination site attI and the gene cassette gene recombination site attC, and connected to The carrier plasmid PUC19 was transformed into Escherichia coli DH5α; real-time fluorescent quantitative PCR was used to measure the number of copies with integrated effect and the copy number without effect, and the integration efficiency was calculated to obtain the activity of integrase. The present invention respectively constructs the carrier with the integrase gene intI and the carrier containing the integrase gene recombination site attI and the gene cassette gene recombination site attC, measures and calculates the size of the integration efficiency in vitro, thereby reflecting the activity of the integrase The size provides a theoretical basis for the study of drug resistance in Escherichia coli.

Description

technical field [0001] The invention relates to the technical field of measuring biological enzyme activity, in particular to a method for measuring drug-resistant Escherichia coli integrase activity. Background technique [0002] Escherichia coli is a common pathogenic bacteria. Due to the widespread and continuous inappropriate use of antibiotics, a large number of resistant strains of E. coli have emerged, making the clinical treatment of E. coli disease very difficult, and sometimes even no curable drug can be found. , Therefore, in recent years, Escherichia coli drug resistance research is a hot spot. [0003] Integron is a mobile DNA molecule with a unique structure that can capture and integrate exogenous genes and transform them into functional gene expression units. Integrons can be located on plasmids, chromosomes or themselves as a component of transposons Part of it is involved in the transfer, and the integron consists of three parts: the 5'conserved end, the 3...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2537/16
Inventor 贾芳杨江流
Owner 河套学院