H5N1亚型禽流感病毒HA部分基因的克隆与可溶性表达

本研究根据Genbank中记录的禽流感H5N1亚型HA基因序列,设计了一对特异性引物,提取禽流感H5N1亚型病毒RNA,并利用一步法RT-PCR方法扩增HA部分基因,与pMD18-T载体连接克隆扩增并鉴定后提取质粒与32a质粒同时分别双酶切并进行连接克隆扩增,提取重组质粒后转入E.coli Rosetta细胞中进行原核表达,通过检测证明得到了可溶性表达,大小约为35kD左右。从而为进一步试验和研究打下了坚实的基础。

Clonlng and Prokaryotic Expression of Partial Gene of HA of H5N1 AIV

The primers were designed by the sequence of the hemagglutinin (HA) gene of avian influenza virus (AIV) subtype H5N1 recorded in the GeneBank.These primers were used to amplify part of the HA gene by one-step RT-PCR through the RNA extracted by subtype H5N1 virus.Then,the PCR product was connected and cloned into the cloning vector pMD18-T.The plasmid which is correct was double digested with the plasmid 32a at the same time and then connected together and cloned.The recombinant plamid was then transformed into Escherichia coli strain Rosetta for prokaryotic expression.The expressed protein is proven to be soluble and about 22 kD.This experiment provided a solid foundation for further experiment and research of AIV gene.