肺炎克雷伯菌榆中分离株OmpK17基因的克隆、生物信息学分析及免疫原性研究

doi:10.16431/j.cnki.1671-7236.2019.05.024

Abstract

Abstract:

This study was aimed to explore the sequence characteristics of OmpK17 gene of Klebsiella pneumonia (K.pneumonia) and its immunogenicity.Aaccording to the OmpK17 gene sequence (accession No.:U52843.1) of K.pneumonia in GenBank,one pair of specific primers was designed,and the sequence of OmpK17 gene CDS of K.pneumonia Yuzhong (YZ) strain was amplified using PCR,the nucleotide and deduced amino acid sequences of OmpK17 gene were analyzed using bioinformatics softwares.The prokaryotic expression vector pET-OmpK17 was constructed and transformed into E.coli BL21(DE3),then the recombinant protein was expressed by IPTG induction.The recombinant protein was purified and the anti-sera against His-OmpK17 was prepared through immunizing rabbit.The immunogenicity of recombinant protein was analyzed by ELISA and Western blotting.The results showed that the sequence of OmpK17 gene of K.pneumonia YZ strain was highly conserved,its CDS sequence was 513 bp in length and encoded 170 amino acids,and the homology of nucleotide sequences of OmpK17 gene was above 99.6% between YZ strain and other K.pneumonia in GenBank.The molecular weight of OmpK17 protein was 18.5 ku,the hydrophilicity was strong,which had 1 signal peptide,1 transmembrane region and 6 potential antigenic determinant regions.Moreover,OmpK17 was a barrel-shaped protein,which suggested that it was a porin protein.In addition,the SDS-PAGE results indicated that the recombinant protein was successfully expressed in E.coli BL21 (DE3),and the expression product was mainly present in the form of inclusion body.The ELISA and Western blotting results showed that the OmpK17 of K.pneumonia had good immunogenicity.This study cloned the sequence of OmpK17 gene of K.pneumonia and expressed its encoded protein which had good immunogenicity.It laid the foundation for the study of the biological characteristics of OmpK17 gene and the establishment of K.pneumoniae detection method,and provided a theoretical basis for the development of genetic engineering vaccine of K.pneumoniae.

Key words: Klebsiella pneumoniae; OmpK17 gene; cloning; bioinformatics; prokaryotic expression; immunogenicity

CLC Number:

S852.61⁺2

CHENG Jiahai, YIN Juanbin, BAI Shanze, TIAN Zeyang, PANG Honghong, BAO Shijun. Cloning,Bioinformatics and Immunogenicity Analysis of OmpK17 Gene of Klebsiella pneumoniae YZ Strain[J]. , 2019, 46(5): 1456-1465.

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Cloning,Bioinformatics and Immunogenicity Analysis of OmpK17 Gene of Klebsiella pneumoniae YZ Strain

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