牛双芽巴贝斯虫新疆株HSP20基因的克隆和真核表达质粒的构建

通过设计牛双芽巴贝斯虫热休克蛋白HSP20基因序列的特异性引物,采用PCR方法从疑似"牛焦虫病"的患牛全血基因组中扩增得到699 bp的HSP20基因,将其连入pMD18-T测序、鉴定,再将验证的699 bp的HSP20基因克隆至真核表达载体pcDNA3.1(+)中,构建真核载体PCDNA3.1-HSP20。经再次测序、鉴定后,尾静脉注射小白鼠进行HSP20基因的瞬时表达,取其肝脏提取总RNA,经RT-PCR方法扩增出一条534bp的目的条带,表明真核表达质粒构建成功。

Cloning the HSP20 Gene of B.bigemina Isolated in Xinjiang and Construction of the Eukaryotic Expression Plasmid

The special primers were designed by the heat shock protein gene sequence of B.bigemina pubulished in GenBank.and a 699 bp HSP20 gene fragment was amplified by PCR from the whole blood genome in the infected cattle.The PCR product was cloned into a pMD18-T vector,then was sequenced and identified.The 699bp HSP20 gene was then cloned into Eukaryotic vector pcDNA3.1(+)to construct a eukaryotic expression plasmid pcDNA3.1-HSP20 before sequencing and identifying again.Following the pcDNA3.1-HSP20 was transiently expressed in mice injected by invena caudalis,the total RNA was abstructed from murine liver and a 534 bp of bright stripe was found by RT-PCR amplification,suggesting the eukaryotic expression plasmid was successfully constructed.