《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (5): 1456-1465.doi: 10.16431/j.cnki.1671-7236.2019.05.024

• 预防兽医 • 上一篇    下一篇

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

程家海, 殷娟斌, 白珊泽, 田泽暘, 庞红红, 包世俊   

  1. 甘肃农业大学动物医学院, 兰州 730070
  • 收稿日期:2018-09-26 出版日期:2019-05-20 发布日期:2019-05-20
  • 通讯作者: 包世俊 E-mail:bsjdy@126.com
  • 作者简介:程家海(1997-),男,内蒙古乌兰察布人,本科,研究方向:动物医学,E-mail:chjh_2015@126.com
  • 基金资助:

    甘肃农业大学省级大学生创新创业训练计划(201710733016);国家自然科学基金(31360620)

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

CHENG Jiahai, YIN Juanbin, BAI Shanze, TIAN Zeyang, PANG Honghong, BAO Shijun   

  1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
  • Received:2018-09-26 Online:2019-05-20 Published:2019-05-20

摘要:

试验旨在研究肺炎克雷伯菌(Klebsiella pneumoniae)外膜蛋白OmpK17基因序列特征及其免疫原性。参照GenBank中肺炎克雷伯菌外膜蛋白OmpK17基因序列(登录号:U52843.1)设计1对特异性引物,应用PCR技术扩增获得了肺炎克雷伯菌榆中分离株(YZ株)OmpK17基因CDS序列,应用相关软件及程序对OmpK17基因核苷酸序列及其推导的氨基酸序列进行生物信息学分析,构建其原核表达载体pET-OmpK17,并在大肠杆菌BL21(DE3)中进行了表达。表达产物纯化后免疫新西兰兔制备多抗血清,应用ELISA和Western blotting分析了重组蛋白的免疫原性。结果显示,肺炎克雷伯菌YZ株外膜蛋白OmpK17基因高度保守,其CDS序列全长513 bp,编码170个氨基酸,与GenBank中其他肺炎克雷伯菌OmpK17基因序列同源性均高于99.6%;OmpK17蛋白分子质量约为18.5 ku,具有较强的亲水性,含有1个信号肽、1个跨膜区和6个抗原决定簇区域,是一种具有桶状三维结构的跨膜蛋白。SDS-PAGE结果显示,OmpK17基因在大肠杆菌中获得表达且表达产物主要以包涵体形式存在。ELISA及Western blotting结果表明,肺炎克雷伯菌OmpK17蛋白具有良好的免疫原性。本研究克隆和表达了肺炎克雷伯菌OmpK17基因及其编码蛋白,并证实该蛋白具有良好的免疫原性,为OmpK17基因的生物学特性研究和肺炎克雷伯菌检测方法的建立奠定了基础,为肺炎克雷伯菌基因工程疫苗的研发提供了理论依据。

关键词: 肺炎克雷伯菌; OmpK17基因; 克隆; 生物信息学; 原核表达; 免疫原性

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

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