表达A型产气荚膜梭菌FBA蛋白的延迟裂解型沙门菌载体的构建  

Preparation of the FBA protein of Clostridium perfringens type A strain and its functional application

作　　者：姜延龙 王占楠 曾庆丰 王棋 姚心茹 王成宇 胡译文 杨桂连 王春凤 JIANG Yan-long, WANG Zhan-nan, ZENG Qing-feng, WANG Qi, YAO Xin-ru, WANG Cheng-yu, HU Yi-wen, YANG Gui-lian, WANG Chun-feng(Jilin Provincial Engineering Research Center of Animal Probiotics/College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118 ,Chin)

机构地区：[1]吉林农业大学动物科学技术学院/吉林省动物微生态制剂工程研究中心

出　　处：《中国兽医学报》2018年第5期906-910,共5页Chinese Journal of Veterinary Science

基　　金：国家“863”计划资助项目(2013AA102806);国家自然科学基金资助项目(31272552,31272541,31672528,31602092);吉林省科技发展计划项目(20160519011JH);吉林省工业创新特殊项目(2016C063)

摘　　要：利用PCR技术扩增FBA基因序列，并将其克隆到表达栽体pET-30a（＋）中，获得pET-30a—FBA重组质粒。将重组质粒转化到BL21感受态细胞中，经IPTG诱导表达His—FBA蛋白，通过His—tagNi柱亲和层析纯化后，免疫新西兰大白兔制备FBA特异性多克隆抗体。再利用PCR技术将FBA基因与表达载体pYA3681连接，构建了表达载体pYA5129，电转至延迟裂解性沙门菌x11802后以制备的FBA多抗为一抗进行免疫印迹试验，验证了其免疫原性；同时比较了不同浓度阿拉伯糖对重组沙门菌生长性能的影响，优化了阿拉伯糖最佳添加浓度，为后续研究其作为肉鸡坏死性肠炎疫苗的可行性奠定基础。The present study was conducted to construct the regulated delayed lysis Salmonella vector expressing fructose 1,6-hiphosphate aldolase （FBA） protein of Clostridium perfringens type A strain. The Fba gene was amplified by PCR approach and coloned into the prokaryotic expression vector pET-30a（＋） to construct the recombinant plasmid pET-30a-FBA. Then, the recombinant plasmid was transformed into the competent E. coli BL21 cells which were then induced by isopropyl-β-thiogalactopyranoside （IPTG）. The recombinant His-FBA fusion protein was purified by a nickel ion metal chelate column . Subsequently,the purified His-FBA protein was used as an immunogen to immunize the New Zealand white rabbits to prepare its polyclonal antibodies. The fba gene was also linked with pYA3681 to construct a plasmid pYA5129 which was designed to express FBA protein in regulated delayed lysis Salmonella. The synthesis of FBA protein in Salmonella was confirmed by Western blot assay using the polyclonal FBA antibody as prime antibody,indicating its possibility to he used in vaccine study.

关 键 词：A型产气荚膜梭菌 FBA 原核表达 多克隆抗体 延迟裂解型沙门菌 

分 类 号：S852.61[农业科学—基础兽医学]