Drug-resistant escherichia coli integrase activity determination method

A technology for the determination of Escherichia coli and activity, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve problems such as multi-drug resistance of bacteria

Active Publication Date: 2018-05-11
河套学院
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  • Description
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  • 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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  • Drug-resistant escherichia coli integrase activity determination 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 invention relates to the technical field of biological enzyme activity determination and particularly relates to a drug-resistant escherichia coli integrase activity determination method. The method comprises extracting plasmid DNA of escherichia coli, carrying out PCR amplification through the plasmid DNA as a template to obtain an integron gene intI for encoding an integrase, connecting theintegron gene intI to an expression vector pEASY-E1, transferring the expression vector pEASY-E1 to escherichia coli BL21(DE3), carrying out PCR amplification to obtain a DNA fragment containing an integrase gene recombination site attI and a gene cassette gene recombination site attC, connecting the DNA fragment to a vector plasmid PUC19, transferring the vector plasmid PUC19 into escherichia coli DH5 alpha, carrying out real-time fluorescence quantitative PCR determination on the copy number with the integration action and the copy number without the integration action, and calculating the integration efficiency to acquire the integrase activity. The method constructs the vector with the integrase integron gene intI and the vector containing the integrase gene recombination site attI andthe gene cassette gene recombination site attC, and measures in vitro and calculates the integration efficiency so as to reflect the integrase activity. The method provides the theoretical basis forthe study of escherichia coli resistance to drugs.

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