牛脂联素基因真核表达载体的构建及鉴定

Construction and identification of eukaryotic expression vector of bovine adiponectin gene

为了构建牛脂联素基因真核表达载体,试验采用RT-PCR方法扩增牛脂联素(bovine adi-ponectin,BovADPN)基因,将其克隆到pMD18-T载体中,测序正确的质粒经EcoRⅠ和NotⅠ双酶切,回收目的基因片段将其定向克隆到pPICZαA载体中,构建重组质粒pPICZαA BovADPN。结果表明:克隆的基因序列与GenBank公布的序列有100%的同源性;目的基因正向插入,阅读框正确无误,说明牛脂联素基因真核表达载体构建成功。

To construct eukaryotic expression vector of bovine adiponeetin gene, the full length cDNA encoding ADPN of bovine was amplified by RT - PCR with the specific primers. Then the gene of ADPN was cloned into the vector T. It was subcloned into the eukaryotic expression vector pPICZαA after the sequence was identified by double restriction endonucleases digestion and sequencing analysis. The results showed that the gene fragment showed 100% identity to ADPN eDNA which had been deposited in the GenBank database, ligation direction of the objective gene fragment was correct and was in agreement with correct reading frame. It showed that eukaryotic expression vector pPICZαA - BovADPN had been constructed successfully.