牛双芽巴贝斯虫热休克蛋白基因外显子的克隆与原核表达

从患牛焦虫病的牛全血样品中抽提总RNA，通过设计特异性引物，经RT-PCR扩增牛双芽巴贝斯虫热休克蛋白20（HSP20）基因的外显子，将其插入pMD18-T载体进行测序、鉴定，构建原核表达质粒载体PGEX-4T-2-HSP20（exon）。试验结果显示牛双芽巴贝斯虫HSP20基因外显子已被成功克隆。该基因的外显子序列与国际标准株HSP20外显子的序列存在两个碱基的突变，导致其编码的蛋白质出现了一个氨基酸的变异。测序结果还表明，克隆出的牛双芽巴贝斯虫HSP20基因外显子已正向插入原核质粒表达载体PGEX-4T-2，成功构建了PGEX-4T-2-HSP20（exon）表达载体，并在大肠杆菌BL21（DE3）中的得到表达。

Cloning Exons of the Heat Shock Protein Gene of B.bigemina and the Construction of Recombined Prokaryotic Expression Plasmid

The special primers were designed by exons of the heat shock protein gene sequence of B. bigemina pubulished in GenBank. The exons of the heat shock protein gene was amplified by RT-PCR from the tatol RNA abstructed from whole blood in the infected cattle and was cloned into a pMD18-T vector before sequencing and identification. A prokaryotic expression vector PGEX-4T-2-HSP20（exon） was then constructed. The result was shown that exons of the heat shock protein gene sequence of B. bigemina was successfully cloned. There were two mutant base pairs among the exons of the heat shock protein gene sequence,comparing that of standard gene published in Genbank, resulting in a mutant of amino acid in the coded protein. The result of sequencing was also shown that cloned exons of the heat shock protein gene sequence of B. bigemina was ligated into the prokaryotic expression vector PGEX-4T-2 to constructed PGEX-4T-2-HSP20 （exon） successfully. The protein was expressed in the E. coil BL21（DE3）.