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Integron In1085

An integron, e.colijm109 technology, used in DNA preparation, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., to prevent outbreaks and reduce selection pressure.

Inactive Publication Date: 2015-09-02
王冬国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibacterial drugs have played an important role in controlling Escherichia coli infection, but with the widespread use of antibacterial drugs, drug-resistant strains of Escherichia coli, especially multi-drug-resistant strains, continue to appear. important factor for industry development

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Identification of integron In1085

[0028] 1. Isolation and identification of Eco336 strain

[0029] 1.1 Materials

[0030] Bacterial susceptibility card: AST-GN13 from bioMerieux, France. AST-GN13 drug-sensitive types include: amikacin, ampicillin, ampicillin / sulbactam, aztreonam, cefazolin, cefepime, cefotetan, ceftazidime, ceftriaxone, ciproxa Star, ertapenem, gentamicin, imipenem, levofloxacin, nitrofurantoin, piperacillin / tazobactam, tobramycin, SMZ.

[0031] Supplementary drug-sensitivity discs (drug-sensitivity plate agar diffusion test): the discs are from Oxoid Company in the United Kingdom, with cefoperazone / sulbactam (75 μg / 30 μg), cotrimoxazole (30 μg), and chloramphenicol (30 μg) .

[0032] 1.2 Method

[0033] Apparatus identification: transfer positive bacterial strains from the urine culture of patients in the Urology Department of Taizhou Municipal Hospital to blood plates for isolation and culture (at 35°C containing 5% CO 2 Cultivate in...

Embodiment 2

[0054] Example 2 Plasmid transduction experiments to study the function of integron In1085

[0055] 1. Method

[0056] (1) The donor strain is Eco336 strain, and the recipient strain is E.coli J53 Az R (resistant to sodium azide). The donor bacteria and recipient bacteria were inoculated on LB plates, respectively, and cultured overnight at 35°C. Pick a single colony and inoculate them in 4 mL of LB broth, and culture at 37°C and 220 r / min until the logarithmic growth phase. Take 0.5 mL of donor and recipient bacteria in 4 mL of LB broth, and culture overnight at 37°C. Zygotes were screened on trypan soy agar (TSA) plates containing sodium azide (300 mg / L) and amifloxacin (0.03 mg / L). Incubate at 35°C for 18-24h. The plasmid of the drug-resistant strain was extracted (the steps are the same as in Example 1), and the sequence of the integron In1085 was detected by the PCR method.

[0057] The primers for amplifying the integron In1085 sequence are shown in Table 1:

[00...

Embodiment 3

[0068] Example 3 Gene recombination experiments to study the function of integron In1085

[0069] 1. Method

[0070] (1) Ligate the PCR product in Example 2 (the nucleotide sequence is SEQ ID NO.1) to the pMD18T vector, add 100 μl of E.coli JM109 to the competent state of the ligated product, and transform it in a cell containing IPTG, x-gal, Cultivate on the Amp plate, extract plasmids from IPTG, x-gal, and Amp drug-resistant strains and sequence them (the steps are the same as in Example 1), and detect whether the integron In1085 is inserted into the genome. Then the integron In1085 sequence was cloned into the pET32a vector, the insertion site of the integron was at the BamHI restriction site on the pET32a vector, prepared according to conventional methods, and the pET32a recombinant plasmid containing the integron In1085 sequence was obtained. Transform the recombinant plasmid into competent E.coli JM109 cells.

[0071] (2) Detection of drug resistance of successfully co...

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Abstract

The invention discloses an integron In1085 of which the sequence is shown in SEQ ID NO.1. The integron In1085 is discovered in the genome of a drug-resistant escherichia coli Eco336. The discovery of the integron In1085 has important theoretical and practical significance in study on the molecular mechanism of bacterial drug resistance transfer at a gene level, further provides a certain guidance effect for clinical medication and is beneficial to reduction of clinical use of some antibacterial medicines, reduction of the selection pressure of horizontal transfer of drug resistance gene cassettes and prevention of outbreak of drug-resistant bacteria.

Description

technical field [0001] The invention belongs to the field of molecular biology technology and drug-resistant bacteria monitoring, and relates to a newly discovered integron closely related to drug resistance of Escherichia coli. Background technique [0002] Escherichia coli is one of the most common pathogenic bacteria in medicine and veterinary clinics, which has caused serious damage to human health and the development of animal husbandry. Antibacterial drugs have played an important role in controlling Escherichia coli infection, but with the widespread use of antibacterial drugs, drug-resistant strains of Escherichia coli, especially multi-drug-resistant strains, continue to appear. important factor for industry development. [0003] Integron (integron) is a recombinant expression system that can recognize, capture, integrate or cut extracellular episomal genes or gene fragments through its own encoded integrase, and transform them into functional genes. Integrons can...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N15/10
Inventor 王冬国陈佳玉陶宝鸿牟永华李海军
Owner 王冬国
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