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Novel drug resistant gene of Klebsiella pneumoniae

A technology of drug-resistant strains and amino acids, applied in antibacterial drugs, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2014-09-10
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports of pneumonia caused by gram-negative bacteria (GNB) other than Klebsiella

Method used

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  • Novel drug resistant gene of Klebsiella pneumoniae
  • Novel drug resistant gene of Klebsiella pneumoniae
  • Novel drug resistant gene of Klebsiella pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Discovery of New Resistance Gene KPC-15

[0026] 1. Materials and methods

[0027] 1. Materials

[0028] VITEK2 from bioMerieux, France; Bacterial identification card; VITEK2GN Test kit; Bacterial susceptibility card; prime); TakaRa’s miniBEST plasmid purification Kit; high-fidelity polymerase, and other reagents are commercially available.

[0029] 2. Method

[0030] 2.1 Isolation and identification of Klebsiella pneumoniae KP1241 strain

[0031] Transfer the positive strains from the blood culture of the cardiothoracic surgery patients in Taizhou Municipal Hospital to the blood plate for isolation and culture, and culture them in an incubator containing 5% CO2 at 35°C for 16-18 hours, and then use the VITEK2 of bioMerieux to isolate the isolated monoclonal strains according to the operating procedures. Bacteria identification and drug susceptibility analysis system and drug sensitivity plate agar diffusion method (supplement drug sensitivity paper sheet) w...

Embodiment 2

[0068] Example 2 Bacterial plasmid transduction experiment and drug resistance detection

[0069] 1. Materials and methods

[0070] 1. Materials

[0071] Klebsiella pneumoniae (accession number KP1241); wild-type E. coli J53.

[0072] 2. Methods and results

[0073] Klebsiella pneumoniae KP1241 and wild-type E.coli J53AzR (resistance to sodium azide) were used as donor bacteria and recipient bacteria respectively, and 0.5ml of each in the logarithmic growth phase was added to 4ml of fresh LB Incubate overnight in broth. The zygotes were screened on trypan soy agar (TSA) plates containing sodium azide (300mg / L) and ciprofloxacin (0.03mg / L), and incubated at 35°C for 18-24 hours. The screened zygotes were analyzed for their sensitivity to drugs with the VITEK2 bacterial identification and drug sensitivity analysis system of bioMerieux and the drug sensitivity plate agar diffusion method (supplementary drug sensitivity paper). The results are shown in Table 2:

[0074] Table...

Embodiment 3

[0078] The construction of embodiment 3 constitutive KPC-15 gene expression bacteria

[0079] 1. Materials and methods

[0080] 1. Materials

[0081] E.coli JM109; pMD18T carrier of TaKaRa Company; pBR322 carrier; restriction endonuclease Pst I and EcoR I of TaKaRa Company; agarose gel recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.; T4 DNA ligase.

[0082] 2. Method

[0083] 2.1 Construction of constitutive KPC-15 gene expression strain E.coli JM109 (pET32a-KPC-15)

[0084] The PCR product in Example 1 was ligated with the pMD18T vector, and the ligated product was added to 100 μl JM109 competent for transformation, cultured on a plate containing IPTG, x-gal, and Amp, and sequenced to verify that the gene sequence and its gene insertion direction were correct. Then the KPC-15 gene was cloned into the pET32a vector to obtain the pET32a-KPC-15 plasmid containing the KPC-15 gene. The recombinant plasmid was transformed into competent E.coli JM109 cells, c...

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Abstract

The invention discloses a novel drug resistant gene of Klebsiella pneumonia. The novel drug resistant gene of the Klebsiella pneumonia is obtained by extracting the Klebsiella pneumonia which is drug resistant against imipenem and ertapenem. The nucleotide sequence of the novel drug resistant gene of the Klebsiella pneumonia is SEQ ID NO. 1 and the amino acid sequence is SEQ ID NO. 2. Functions of the novel gene are researched through a plasmid transduction experiment and a prokaryotic expression experiment, and a result shows that the novel gene is drug-resistant against a plurality of drugs. The novel drug resistant gene of the pneumonia Klebsiella has a great significance on reasonable use of antibiotics, reduction of carbapenems drug-resistant bacteria produced during severe infection, and research and development of new drugs for inhibiting drug-resistance performance of bacteria.

Description

technical field [0001] The present invention relates to a new drug-resistant gene of Klebsiella pneumoniae, in particular, the present invention relates to a new drug-resistant gene found in drug-resistant Klebsiella pneumoniae isolated from a patient's blood culture specimen and The protein encoded by this gene has been confirmed to play an important role in bacterial drug resistance. Background technique [0002] Klebsiella is a rod-shaped Gram-negative bacterium, usually covered by a capsule formed by a large number of viscous polysaccharides. Among them, Klebsiella pneumoniae is highly pathogenic to humans and is an important conditional pathogen and One of hospital-acquired infections. [0003] Klebsiella pneumoniae (Klebsiella pneumoniae), also known as Klebsiella pneumoniae or Friedlander bacillus, is the earliest known gram-negative bacillus that can cause pneumonia. The shape of the bacteria is short and thick bacilli, the size is 0.5-0.8×(1-2) μm, arranged alone,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/26C12N15/31C12N1/21A61K38/16A61P31/04
Inventor 王冬国杨林军牟永华侯伟伍海建张瑾
Owner ACROBIOSYSTEMS INC
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