摘要
根据Ⅰ型鸭甲型肝炎病毒(Duck Hepatitis A Virus type-Ⅰ,DHAV-Ⅰ)SH株VP0基因序列设计1对特异性引物,RT-PCR方法扩增出VP0基因,克隆入原核表达载体p ET-32a,转化大肠杆菌BL21(DE3),在IPTG的诱导下重组蛋白获得了成功表达。SDS-PAGE结果显示,表达的重组蛋白相对分子量约为48 k D,Western-blotting分析表明该蛋白能与His组氨酸单抗发生特异性反应。将目的蛋白切胶纯化后免疫ICR小鼠,制备了针对重组蛋白的多抗血清,Western-blotting分析证明制备的抗血清能够与DHAV-Ⅰ感染的SPF鸡胚尿囊液总蛋白发生特异反应。以上结果表明,VP0基因在大肠杆菌中获得了成功表达,且制备的多抗血清可以用于VP0蛋白的检测。
According to the genome sequences of duck hepatitis A virus type-Ⅰ（ DHAV-Ⅰ）,one pair of specific primers was designed. The VP0 genes were amplified by RT-PCR,and then were cloned into prokaryotic expression vector p ET-32 a,and the recombinant plasmids were transformed into BL21（ DE3） E. coli. Then the recombinant proteins were successfully expressed following IPTG induction.SDS-PAGE showed that the approximate molecular weight of recombinant expression proteins was 48 k D. Western blotting assay revealed that the desired proteins could be recognized by the monoclonal antibody against histidine-tagged proteins. The antiserum against the recombinant proteins were produced by immunized ICR mouse with recombinant proteins. Western-blotting results showed that the antiserum could react specifically with DHAV-Ⅰ allantoic fluid. These results showed that VP0 gene was successfully expressed in E. coli,and the antiserum could be used for detection of VP0 proteins.
出处
《河南农业大学学报》
CAS
CSCD
北大核心
2015年第5期658-661,共4页
Journal of Henan Agricultural University
基金
国家自然科学基金项目(31302096)
江苏省农业支撑项目(BE2013415)
江苏省六大人才高峰项目(NY-009)
