鸭源鸡杆菌flfA基因的克隆表达及抗原性分析  

Prokaryotic expression of fimbrial protein FlfA of Gallibacterium anatis and its antigenic activity

作　　者：彭志锋[1,2] 张真真 卢彩景[1] 陈陆 赵军[1] 王川庆[1] 王继洋[1] 王坤芃[1] 杨霞 PENG Zhi-feng1,2 , ZHANG Zhen-zhen1 , LU Cai-jing1, CHEN Lu1, ZHAO Jun1, WANG Chuan-qing1, WANG Ji-yang1 ,WANG Kun-peng1 ,YANG Xia1(1. Institute of Poultry Diseases, Henan Agricultural University, Zhengzhou 450002, China; 2. College of Animal Medicine, Henan University of Animal Husbandry and Economy ,Zhengzhou 450046 ,Chin)

机构地区：[1]河南农业大学禽病研究所,河南郑州450002 [2]河南牧业经济学院动物医学院,河南郑州450046

出　　处：《中国兽医学报》2018年第5期900-905,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(U1404328);河南省自然科学基金资助项目(162300410153);河南省高校科技创新团队与支持计划资助项目(14IRTSHN015)

摘　　要：用PCR方法从鸭源鸡杆菌中国分离株PDS-RZ-1-SLG中扩增flfA基因，并运用生物信息学软件分析鸭源鸡杆菌菌毛蛋白FlfA的二级结构、表面可及性与B细胞优势表位簇；将目的基因插入原核表达载体Pet28a（＋），经PCR、酶切及测序鉴定成功构建重组表达质粒pET28a（＋）-FlfA，然后转化到大肠杆菌BL21，IPTG诱导重组质粒表达；SDS-PAGE和Western blot鉴定FlfA的表达及抗原性。结果显示：成功获得与预期大小一致的573bp的flfA基因；FlfA蛋白结构分析显示其二级结构以无规则卷曲为主，表面存在很多抗原表位；PCR及酶切等鉴定表明重组表达质粒pET28a（＋）-FlfA构建成功。SDS-PAGE分析显示，经IPTG诱导表达获得相对分子质量约20000的重组蛋白；Western blot分析结果显示重组菌毛蛋白能被兔抗rFlfA多抗血清和鸡抗鸭源鸡杆菌血清识别，抗原性良好；而且，rFlfA多抗血清与菌毛蛋白具有良好反应性。This study aimed at the analysis of antigenic activity of fimbrial protein FlfA of Gallibacterium anatis （G. anatis） so as to further explore the prevention measures. To express flfA gene of G. anatis by prokaryotic expression system and to identify its antigenicity, intact flfA gene of G. anatis PDS-RU-1-SLG isolated in China was amplified by PCR. Then, the second structure, ac- cessibility and antigenicity of fimbrial protein FlfA of G. anatis were predicted by bioinformatics software. Recombiaant prokaryotic expression plasmid pET28a （＋） with FlfA protein gene was constructed. The correct plasmid was confirmed by the restriction enzymes digestion and sequencing. Then the recombinant plasmids were transformed into E. coli BL21 ,and the bacteria were induced by IPTG. The expression and immune reactivity of FlfA protein were detected by SDSPAGE and Western blot, respectively. The results indicated that expected fifA genes was acquied by PCR amplification and sequencing;FlfA protein was composed of random curl mainly and possessed a plenty of epitopes. The results of SDS-PAGE revealed that the relative molecular weight of rFlfA protein was consistent with desired results. Western blot showed that rFlfA can be strongly reconnized by rabbit serum immunized by recombinant FlfA protein,and had good immunogenicity. Moreover,the FlfA proteins from different G. anatis strains had good reactivity to antirFlfA serum,which laid a solid found for the further study of biological function of FlfA protein and preventive strategies for G. anatis infection.

关 键 词：鸭源鸡杆菌 flfA基因 基因克隆 原核表达 抗原性 

分 类 号：S852.61[农业科学—基础兽医学]