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Integron In1069

An integron, EC6335 technology, used in DNA preparation, introduction of foreign genetic material and microorganisms using vectors, etc., can solve problems such as multi-drug resistance, animal husbandry and human health hazards, and achieve the effect of preventing outbreaks and reducing selection pressure.

Inactive Publication Date: 2015-03-25
王冬国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the widespread, continuous and improper application of antibiotics, the drug resistance spectrum of Escherichia coli continues to expand and the level of drug resistance continues to increase. The phenomenon of drug resistance and multi-drug resistance of E. the harm of

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Identification of integron In1069

[0033] 1. Isolation and identification of strain EC6335

[0034] 1.1 Materials

[0035] Bacterial susceptibility card: AST-GN13 from bio Merieux, 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.

[0036] Supplementary drug-sensitivity disc (drug-sensitivity plate agar diffusion test): the disc comes from Oxoid Company in the United Kingdom, with cefoperazone / sulbactam (75 μg / 30 μg).

[0037] 1.2 Method

[0038] Apparatus identification: transfer positive bacterial strains from patients in the intensive care unit of Taizhou Municipal Hospital to blood plates for isolation and culture (at 35°C containing 5% CO 2 Cultivate in the incubator for 16-18h), and then carry out bacterial identification a...

Embodiment 2

[0054] Example 2 Plasmid transduction experiment to study the function of integron In1069

[0055] 1. Method

[0056] (1) The donor bacterium is EC6335 strain, and the recipient bacterium is E.coli J53AzR (resistance 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 0.5ml of donor and recipient bacteria in 4ml 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 imipenem (2 mg / L). Incubate at 35°C for 18-24h. Extract the plasmid of drug-resistant bacterial strain (step is the same as embodiment 1) be PCR amplification template, use the primer detection intI1, tnpA, aacA4 '-3, *RIP, Orf44, IMP-1, TniC gene fragment to exist in Table 1.

[0057] (2) Select positive bacteria...

Embodiment 3

[0060] Example 3 Gene recombination experiment to study the function of integron In1069

[0061] 1. Method

[0062] (1) Integron In1069 sequence was synthesized: the integron In1069 sequence was synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0063] (2) Ligate the synthesized integron In1069 to the pMD18T vector, add 100 μl of E.coli JM109 to the competent state of the ligation product, and culture it on a plate containing IPTG, x-gal, and Amp. IPTG, x-gal, and Amp Plasmids were extracted from resistant strains, and whether the integron In1069 was inserted into the genome was detected by PCR (the steps were the same as in Example 2). Then the integron In1069 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 In1069 sequence was obtained. Transform the recombinant plasmid into compe...

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Abstract

The invention discloses an integron In1069 with a sequence as shown in SEQ ID NO.1. The integron is found in a genome of drug-resistant Escherichia coli EC6335. The integron contains a plurality of drug-resistant gene cassettes. Therefore, due to the discovery of the integron disclosed by the invention, a certain guiding effect is provided for clinical medication, some clinically-used antibacterial agents can be favorably reduced, the selective pressure for horizontal transfer of the drug-resistant gene cassettes is reduced, and the outbreak of drug-resistant bacteria is avoided.

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 In1069 closely related to drug resistance of Escherichia coli. Background technique [0002] Escherichia coli is a typical Gram-negative bacillus, and colibacillosis caused by it is a common disease. The main prevention and control measures for colibacillosis are the application of vaccines and antibiotics. However, with the widespread, continuous and improper application of antibiotics, the drug resistance spectrum of Escherichia coli continues to expand and the level of drug resistance continues to increase. The phenomenon of drug resistance and multi-drug resistance of E. hazards. [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 functio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12N15/10C12N15/70C12R1/19C12R1/22
Inventor 王冬国陈佳玉陶宝鸿伍海建
Owner 王冬国
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