银狐生长激素cDNA克隆和原核表达

为了获得重组的银狐生长激素，本试验用Trizol的方法从银狐脑垂体组织中提取总RNA，以mRNA为模板，用RT-PCR方法扩增出生长激素成熟cDNA片段并克隆到载体质粒pMD18-T中；通过含有BamHⅠ酶切位点的特异引物从pMD18-T-fGH中亚克隆出cDNA片段，并将其重组到pPROEX^TMHta质粒中，构建了银狐生长激素原核表达质粒pPROEX^TMHta-fGH。将pPROEX^TMHta-fGH原核表达质粒转化DH5α大肠杆菌，用终浓度1 mmol/L IPTG进行诱导，通过SDS PAGE进行检测。结果表明在转化的大肠杆菌中检测到分子量为26 kD的fGH蛋白的存在，表达量占菌体蛋白总量的35%，并且表达产物以包涵体的形式存在。

Cloning and Prokaryotie Expression of Growth Hormone cDNA from Vulpes Vulpes

To obtain the recombinant vulpes vulpes growth hormone. The mature vulpes vulpes growth hormone mRNA obtained from pituitary was amplified by RT PCR , cloned into vector pMD18-T and subcloned into prokaryotic expression vector pPROEX^TMHta at restriction enzyme sites BamHⅠ and HindⅢ. The recombinant plasmids pMD18-TfGH cDNA and pPROEX^TMHta-fGH cDNA were identified by restriction enzyme digestion, PCR and sequencing. Then we transfer positive recombinant prokaryotic plasmid into E.Coil, and the protein was expressed under the induction of IPTG at the final concentration of 1 mmol/L. The protein expression was measured by SDS-PAGE, and a high level of fGH protein was obvserved at 26 kD which occupied about 35% of total E.coil protein. The recombined fGH protein existed in the form of inclusion bodies.