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Novel integron In1290

An integron, KP5325 technology, applied in the direction of biochemical equipment and methods, applications, botany equipment and methods, etc., can solve the problems of host bacteria reproduction burden and unclearness, and achieve the effect of preventing explosive epidemics and reducing selection pressure

Inactive Publication Date: 2016-11-30
王冬国
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Integrons gain a survival advantage by capturing favorable gene cassettes, such as drug resistance gene cassettes, in some cases, but they may also capture some gene cassettes that are toxic to the host and cause adverse effects, or over-capture gene cassettes and cause a certain burden on the reproduction of host bacteria
Therefore, in the process of capturing foreign gene cassettes, bacterial integrons must have a complete and fine regulatory mechanism, but this mechanism is still unclear

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Identification of integron In1290

[0030] 1. Isolation and identification of KP5325 strain

[0031] 1.1 Materials

[0032] 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.

[0033] Supplementary drug-sensitivity discs (drug-sensitivity plate agar diffusion test): the discs were from Oxoid, UK, and included co-trimoxazole (30 μg), chloramphenicol (30 μg) and rifampicin (5 μg).

[0034] 1.2 Method

[0035] Apparatus identification: transfer positive bacterial strains from the blood culture of patients in the Department of Infectious Diseases of Taizhou Municipal Hospital to the blood plate for isolation and culture (at 35°C with 5% CO 2 Cultivate in the incubator for 16-18h...

Embodiment 2

[0056] Example 2 Plasmid transduction experiment to study the function of integron In1290

[0057] 1. Method

[0058] (1) The donor bacterium is KP5325 strain, and the recipient bacterium 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 (0.06 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 In1290 sequence of the integron was detected by the PCR method.

[0059] The primers for amplifying the integron In1290 sequence are shown in Table 1: ...

Embodiment 3

[0070] Example 3 Gene recombination experiments to study the function of integron In1290

[0071] 1. Method

[0072] (1) Ligate the PCR product in Example 2 (the nucleotide sequence is SEQ ID NO.1) to the pMD19-T vector, add 100 μL of E.coli JM109 to the competent state of the ligation product, and transform it in a culture medium containing IPTG, x- The gal and Amp plates were cultured, the IPTG, x-gal, and Amp resistant strains were extracted and the plasmids were sequenced (the steps were the same as in Example 1), and it was detected whether the integron In1290 was inserted into the genome. Then the integron In1290 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 In1290 sequence was obtained. Transform the recombinant plasmid into competent E.coli JM109 cells.

[0073] (2) Detection of...

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Abstract

The present invention discloses a novel integron In1290, which has a sequence represented by SEQ ID NO.1. According to the present invention, the integron is obtained by carry out restriction enzyme digestion sequencing identification in a Klebsiella Pneumoniae resistant strain; and the integron contains a plurality of drug resistant gene cassettes, such that the discovery of the integron provides the guidance for the clinical medication, the use of the antibacterial drugs is easily reduced in the clinic, the selection pressure of the horizontal transferring of the drug resistant gene cassettes can be reduced, and the explosive epidemic of the drug-resistant bacteria can be prevented.

Description

technical field [0001] The invention belongs to the fields of molecular biology technology and drug-resistant bacteria monitoring, and relates to a newly discovered integron closely related to the drug resistance of Klebsiella pneumoniae. Background technique [0002] With the advancement of medical level, a wide variety of antibiotics are increasingly being developed and put on the market. It is widely used in clinical and veterinary medicine, and even in animal husbandry, it is often used to promote growth. It is the widespread use of these antibiotics, especially the irrational use, which has caused the problem of bacterial resistance and multi-drug resistance to become more and more serious. Mechanisms are becoming increasingly complex. It is expected that gene mutations may lead to the emergence of strains resistant to a single antibiotic, but it is difficult to explain the resistance to multiple antibiotics in the same strain. Studies have shown that the horizontal t...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C12N1/21C12N15/03C12N15/10C12R1/19C12R1/22
CPCC07K14/26C12N15/03C12N15/102C12N15/70C12Q2521/301
Inventor 王冬国周冬生陈佳玉
Owner 王冬国
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