滩羊肝脏α-TTP基因克隆及原核表达载体的构建

Cloning of Tan Sheep liver alpha TTP gene and its original nuclear expression vector construction

为克隆滩羊肝脏α-生育酚转移蛋白(α-TTP)CDS区基因并构建其原核表达载体,通过Trizol法提取滩羊肝脏组织总RNA,将其反转录为cDNA后利用人工合成和PCR扩增相结合的方法得到α-TTP CDS区基因,并将其克隆至原核表达载体pET28a,命名为pET28a-TTPA。结果表明:扩增了滩羊肝脏α-TTP CDS区基因,同已公布的绵羊α-TTP序列同源性达99.76%,并且将其成功克隆至原核表达载体pET28a。结果为后续α-TTP基因原核表达及其抗体的制备奠定了基础。

The aim of this study was to clone the CDS area of α-TTP of Tan Sheep and constructed its original nuclear expression vector. RNA of theTan Sheep liver was extracted through Trizol method,and it was reverse transcripted to cDNA. The CDS area of α-TTP was got through synthetic method and PCR amplify method,and it was eventually cloned to original nuclear expression vector pET28a, and named pET28a-TTPA. The results showed that, the CDS area of α-TTP was not only amplified and cloned to the original nuclear expression vector pET28a successfully, but alsoshowed a 99.76% sequence homology with the published sheep α-TTP gene. These results established the foundation for α-TTP prokaryotic expression and the preparation of its antibody.