摘要
为了研究重组鸡传染性支气管炎病毒核蛋白能否作为群特异性诊断抗原加以应用,将鸡传染性支气管炎病毒国内分离株IBV-LX4核蛋白基因亚克隆于大肠杆菌原核表达载体pPROEXTMHT中,构建拟表达重组鸡传染性支气管炎病毒核蛋白的重组质粒pPROEXTMHT-N。经核苷酸序列测定后,阳性质粒转化大肠杆菌DH5α,并进行诱导表达。表达产物经SDS-PAGE、Westernblot鉴定,表明核蛋白在大肠杆菌中获得了表达,表达产物是分子量分别为约56kD和45kD的蛋白,其中分子量为56kD的蛋白表达量约为菌体总蛋白的13%。包涵体被6mol盐酸胍裂解后,通过镍离子亲和树脂进行了纯化,并用纯化的分子量为56kD的重组蛋白免疫新西兰白兔,所获得的兔抗鸡传染性支气管炎病毒核蛋白多克隆抗体,分别与不同致病型的鸡传染性支气管炎病毒进行琼脂扩散反应,结果表明,该多克隆抗体可与各不同致病型毒株发生反应。这初步表明重组鸡传染性支气管炎病毒核蛋白,可作为群特异性诊断抗原用于该病毒的诊断中。
The nucleocapsid gene of infectious bronchitis virus(IBV)st rain LX4 isolated from China was subcl oned into Eco RI/HindⅢ sites of pP ROEX^(TM)HT vector to construct recom binant plasmid pPROEX^(TM)HT-N.The re combinant plasmid was transformed into E.coli DH5α and the bacteria was induced with IPTG.It was demon strated by SDS-PAGE and Western bl ot that two proteins,56kD and 45k D,were expressed in E.coli DH5α.O ne of the protein,56kD,was compar able in size to native nucleocapsi d protein and the other appeared t o be degradated product of the nuc leocapsid protein.The 56kD protei n was purified and used to prepare the antibody against IBV nucleocap sid protein.The results showed that the antibody could react with diff erent IBVs,so the recombinant nucl eocapsid protein is a suitable sou rce of antigen for detection of IB V in chicken.
出处
《病毒学报》
CAS
CSCD
北大核心
2003年第4期342-347,共6页
Chinese Journal of Virology
基金
黑龙江省政府博士后科研启动金资助项目(LRB-KY01045)
关键词
传染性支气管炎病毒
重组核蛋白
多克隆抗体
抗原性
infectious bronchitis virus
recombinant nucleocapsid prote in
polyclonal antibody
antigenicity
