坏死梭杆菌120 ku血凝素相关外膜蛋白的原核表达及免疫原性分析

doi:10.16431/j.cnki.1671-7236.2015.11.004

›› 2015, Vol. 42 ›› Issue (11): 2843-2849.doi: 10.16431/j.cnki.1671-7236.2015.11.004

坏死梭杆菌120 ku血凝素相关外膜蛋白的原核表达及免疫原性分析

吴洪超¹, 刘晓颖¹, 冯二凯^1,2, 朱言柱¹, 万洪理², 冯卓¹, 闫喜军¹, 陈立志^1,2

1. 中国农业科学院特产研究所, 长春 130112;
2. 吉林特研生物技术有限责任公司, 长春 130112

收稿日期:2015-05-13 出版日期:2015-11-20 发布日期:2015-11-26
通讯作者: 陈立志
作者简介:吴洪超(1988-),男,河南郑州人,硕士,研究方向:动物传染病及其病原分子流行病学,E-mail:wu2008213@126.com
基金资助:
吉林省科技发展计划项目:牛、鹿腐蹄病防治关键技术研究与产业化示范(20110223);致病性坏死梭杆菌斑马鱼感染模型的建立及白细胞毒素致病机理研究(201205028)

Prokaryotic Expression and Immunogenicity Analysis of Hemagglutinin-related Outer Membrane Protein from F.necrophorum

WU Hong-chao¹, LIU Xiao-ying¹, FENG Er-kai^1,2, ZHU Yan-zhu¹, WAN Hong-li², FENG Zhuo¹, YAN Xi-jun¹, CHEN Li-zhi^1,2

1. Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China;
2. Jilin Teyan Biological Technology Limited Liability Company, Changchun 130112, China

Received:2015-05-13 Online:2015-11-20 Published:2015-11-26

摘要/Abstract

摘要： 为了揭示坏死梭杆菌(F.necrophorum)120 ku血凝素相关外膜蛋白的免疫原性,根据F.necrophorum 120 ku血凝素相关外膜蛋白基因的抗原表位分布情况设计特异性引物,扩增出3个彼此重叠、覆盖整个开放阅读框的基因片段p1、p2、p3,分别构建重组表达质粒pET-28a-p1、pET-32a-p2、pET-32a-p3,在大肠杆菌中进行诱导表达,通过SDS-PAGE电泳和Western blotting分析重组蛋白的表达情况和反应原性;以纯化的重组蛋白为抗原免疫试验兔,分析重组蛋白的免疫原性。结果显示,重组蛋白P1、P2、P3在大肠杆菌中均得到表达,分子质量分别约为48、59、62 ku,能与F.necrophorum阳性的小鼠血清反应,可以刺激试验兔产生特异性抗体,说明F.necrophorum 120 ku血凝素相关外膜蛋白具有良好的免疫原性,为F.necrophorum基因工程亚单位疫苗的研制提供参考依据。

关键词: 坏死梭杆菌; 血凝素相关外膜蛋白; 原核表达; 免疫原性分析

Abstract: In order to analyze the immunogenicity of 120 ku hemagglutinin-related outer membrane protein from F.necrophorum,specific primers were designed to amplify the 3 truncated overlapping gene fragments covering the whole open reading frame (ORF) based on the result of antigenic epitope analysis.The gene fragments were ligated into the prokaryotic expression vector to construct pET-28a-p1,pET-32a-p2 and pET-32a-p3,respectively.Then recombinant plasmid was induced expression in E.coli.The SDS-PAGE results showed that the expression of recombinant proteins P1,P2 and P3,molecular mass of the expressed proteins were about 48,59 and 62 ku,respectively.Western blotting indicated that the recombinant proteins could be recognized by antiserum against F.necrophorum from mouse.Humoral immunity response against recombinant proteins was detected in the immunized rabbits.Altogether,these findings suggested that 120 ku hemagglutinin-related outer membrane protein from F.necrophorum had good immunogenicity,and this study provided a reference for further research on genetic engineering subunit vaccine of F.necrophorum.

Key words: F.necrophorum; hemagglutinin-related outer membrane protein; prokaryotic expression; immunogenicity analysis

中图分类号:

Q786

吴洪超, 刘晓颖, 冯二凯, 朱言柱, 万洪理, 冯卓, 闫喜军, 陈立志. 坏死梭杆菌120 ku血凝素相关外膜蛋白的原核表达及免疫原性分析[J]. , 2015, 42(11): 2843-2849.

WU Hong-chao, LIU Xiao-ying, FENG Er-kai, ZHU Yan-zhu, WAN Hong-li, FENG Zhuo, YAN Xi-jun, CHEN Li-zhi. Prokaryotic Expression and Immunogenicity Analysis of Hemagglutinin-related Outer Membrane Protein from F.necrophorum[J]. , 2015, 42(11): 2843-2849.

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坏死梭杆菌120 ku血凝素相关外膜蛋白的原核表达及免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of Hemagglutinin-related Outer Membrane Protein from F.necrophorum

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