牛多杀性巴氏杆菌外膜蛋白H的表达与纯化

›› 2013, Vol. 40 ›› Issue (11): 38-41.

牛多杀性巴氏杆菌外膜蛋白H的表达与纯化

胡旭, 梁宏儒, 姜东君, 赵达, 高佳滨, 陈为宏, 尹辉, 乔波, 朱战波

黑龙江八一农垦大学动物科技学院, 黑龙江大庆 163319

收稿日期:2013-04-23 出版日期:2013-11-20 发布日期:2013-12-19
通讯作者: 朱战波 E-mail:zhanbozhu@163.com
作者简介:胡旭(1986-),男,黑龙江人,硕士生,研究方向:家畜传染病诊断与防治。
基金资助:
科技部项目(2012BAD12B05);黑龙江省科技厅重大科技攻关项目(GA09B301-2)。

Expression and Purification of Outer Membrane Protein H of P. multocida

HU Xu, LIANG Hong-ru, JIANG Dong-jun, ZHAO Da, GAO Jia-bin, CHEN Wei-hong, YIN Hui, QIAO Bo, ZHU Zhan-bo

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Received:2013-04-23 Online:2013-11-20 Published:2013-12-19

摘要/Abstract

摘要： 试验对多杀性巴氏杆菌外膜蛋白H(OmpH)基因进行克隆、鉴定,并在原核系统中表达。以多杀性巴氏杆菌(CVCC448)强毒株基因组为模板,扩增OmpH基因,连接T载体,经测序鉴定正确后与表达载体pET-28a连接构建重组表达质粒OmpH-pET28a,将此重组质粒转化入表达宿主E.coli BL21菌株内,抽提质粒,酶切鉴定正确后对转化菌株以IPTG进行诱导,表达产物通过镍离子亲和层析纯化,之后进行SDS-PAGE和Western blotting分析。结果显示,OmpH基因的编码区为978 bp,编码326 个氨基酸残基,融合蛋白分子质量约为37 ku。Western blotting检测结果显示,表达的重组蛋白OmpH可与鼠抗多杀性巴氏杆菌全菌体多抗血清反应得到清晰的目的条带,表明表达的重组蛋白具有良好的免疫原性。多杀性巴氏杆菌OmpH基因的成功表达,为进一步研究其免疫作用奠定了基础。

关键词: 多杀性巴氏杆菌; 外膜蛋白H; 克隆; 表达; 鉴定

Abstract: In this assay,cloning, expression and identification of outer membrane protein H (OmpH) of Pasteurella multocida were conducted to provide the foundation for study of immune effects of OmpH. OmpH gene was amplified from the genomic DNA of P.multocida CVCC448 by using PCR. After being cloned into pMD18-T, OmpH-pMD18-T was sequenced to confirm its identity, and then cloned into expression vector pET-28a. The constructed recombinant plasmid OmpH-pET28a was transformed into E.coli BL21 and induced with IPTG, and the expressed product was identified by SDS-PAGE and Western blotting. Sequence analysis showed that the full coding length of OmpH was 978 bp, which could encode 326 amino acids, expressed recombinant protein was 37 ku. Western blotting result showed that recombinant OmpH could react with sera of rats immunized with P.multocida, which showed that the recombinant protein had good immunogenicities. OmpH-pET28a was constructed successfully, and fusion protein was expressed in E.coli BL21,which laid the foundation for the study of immune effects of OmpH.

Key words: Pasteurella multocida; outer membrane protein H; clone; expression; identification

中图分类号:

S855.1

胡旭, 梁宏儒, 姜东君, 赵达, 高佳滨, 陈为宏, 尹辉, 乔波, 朱战波. 牛多杀性巴氏杆菌外膜蛋白H的表达与纯化[J]. , 2013, 40(11): 38-41.

HU Xu, LIANG Hong-ru, JIANG Dong-jun, ZHAO Da, GAO Jia-bin, CHEN Wei-hong, YIN Hui, QIAO Bo, ZHU Zhan-bo. Expression and Purification of Outer Membrane Protein H of P. multocida[J]. , 2013, 40(11): 38-41.

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