Cloning and Prokaryotic Expression of VP3 Gene of Duck Hepatitis A Virus Type 1 Subtype
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摘要: 参照鸭1型甲肝病毒亚型(DHAV-1a)的基因组序列设计了1对VP3基因特异性扩增引物,通过RT-PCR方法扩增获得了DHAV-1a结构蛋白VP3基因,将纯化的VP3基因与pEASYTM-Blunt Zero Cloning Vector连接,构建DHAV-1aVP3基因克隆重组质粒,然后将VP3基因片段插入pET-32a(+)表达载体,转化E.coli BL21(DE3)感受态细胞。以1.0mmol·L-1 IPTG于37℃诱导表达,经SDS-PAGE和Western blot分析表明VP3基因于大肠杆菌中成功表达,其分子量为47kDa,且表达的VP3蛋白能够与Anti-His Mouse mAb发生特异性反应,具有良好的生物活性,为进一步研究DHAV-1aVP3蛋白功能和以VP3蛋白为抗原研制DHAV-1a诊断试剂盒奠定基础。Abstract: The VP3 gene of the hepatitis A virus type 1subtype(DHAV-1a)in ducks was amplified by the reverse transcription-polymerase chain reaction(RT-PCR)using one pair of specific primers designed according to the published sequences of DHAV-1a.The target DNA was purified and cloned into pEASYTM-Blunt Zero Cloning Vector.The recombinant expression plasmid,pET-32a-VP3,was constructed by inserting the target gene fragment into pET-32a(+)vector and transformed into Escherichia coli BL21(DE3)competent cells.In this study,SDSPAGE and Western-blot analyses showed that the recombinant protein VP3,approximately 47 kDa in molecular mass,was expressed highly in E.coli after pET-32a-VP3 was induced with 1.0mmol·L-1 IPTG at 37℃.The expressed recombinant protein was recognized specifically by Anti-His Mouse mAb showing agood bioactivity.
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Keywords:
- duck hepatitis A virus type 1a /
- VP3gene /
- cloning /
- prokaryotic expression
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