摘要
[目的]用大肠杆菌表达出CIAV的衣壳蛋白(VP1)作为免疫抗原,以建立鸡传染性贫血病毒(CIAV)的血清学诊断方法。[方法]根据已发表的CIAV的衣壳蛋白(VP1)序列,结合Protean软件预测出抗原富集区域,设计并合成1对引物,利用PCR扩增该区域基因,将扩增成功的VP1基因片段经T4连接酶连接至PGEX-6P-1表达载体。连接成功的重组质粒转化不同的大肠杆菌表达菌株,通过IPTG诱导进行原核表达,筛选出表达量最高的菌株及最佳诱导时间和IPTG诱导浓度。以感染CIAV的鸡阳性血清为一抗,对重组蛋白GST-VP1进行Western-blot分析。[结果]成功获得了重组质粒PGEX-VP1,诱导表达后经SDS-PAGE和Western-blot分析,分子量约71k D的融合蛋白GST-VP1在大肠杆菌Rosseta中以包涵体形式大量表达,并能与鸡传染性贫血病毒阳性血清发生反应。[结论]该研究可为抗CIAV单克隆抗体的制备及血清学诊断方法的建立等研究奠定基础。
[ Objective] The research aimed to use capsid protein （VP1） of chicken infectious anemia virus （CIAV） expressed by Escherichia coli as immunizing antigen and establish the serological diagnosis method of CIAV. [ Method ] According to the published VP1 sequence of CIAV, the antigen-enriched regions were predicted by using Protean software. A pair of primers were designed and synthesized for the amplification of targeted VP1 genes by PCR. The amplified fragment of VP1 genes was connected to PGEX-6P-1 expression vector by T4 DNA ligase. The recom- binant plasmid after correct connection was used to transform different strains of Escherichia coli for prokaryotic expression by IPTG inducement. The strain with the highest expression amount was screened out, and the best inducement time and the best inducement concentration of IPTG were confirmed. The positive serum o chicken infected with CIAV was used as primary antibody to conduct Western-blotting on recombinant pro- tein GST-VP1. [ Result] The recombinant plasmid PGEX-VP1 was successfully obtained and verified by SDS-PAGE and Western blot. The fusion protein GST-VP1 with the molecular weight of about 71 kD was abundantly expressed in Rosseta of E. coli in the form of inclusion body. And the recombinant protein could react with positive serum of chicken infectious anemia virus. [ Conclusion] The research could lay the foundation for preparing anti-CIAV monoclonal antibody and establishing the serological diagnosis method of CIAV.
出处
《安徽农业科学》
CAS
2015年第28期118-121,共4页
Journal of Anhui Agricultural Sciences
基金
江苏省"六大人才高峰"高层次人才项目(2014-NY-023)
江苏省高校优势学科建设工程资助项目
关键词
鸡传染性贫血病毒
VP1蛋白
原核表达
Chicken infectious anemia virus
VP1 protein
Prokaryotic expression
