人α-乳白蛋白质基因的克隆及其在奶山羊胎儿成纤维细胞中的表达

摘要

采用PCR方法从人类基因组DNA中扩增hα-LA基因,将扩增产物克隆到pMD19-T载体,并进行测序验证.通过双酶切法将测序正确的hα-LA亚克隆至真核表达载体pCEP4,构建真核表达载体pCLA并进行酶切鉴定.用脂质体法将pCLA转染至奶山羊胎儿成纤维细胞中,用RT-PCR法检测细胞中hα-LA基因mRNA的表达,用Western-bloning法检测培养上清液中hα-LA蛋白质的表达.结果显示:克隆的hα-LA基因序列完全正确,pCLA酶切鉴定正确,RT-PCR检测到转染细胞中hα-LA基因mRNA的表达,Western-blotting检测到转染细胞培养上清液中hα-LA蛋白质.表明所克隆的hα-LA基因组DNA能够在奶山羊胎儿成纤维细胞中正确转录和翻译.%ha-LA gene was amplified from human genome DNA by PCR and cloned into pMD19-T for sequencing. Kpn I and BglⅡ were used to sub-clone ha-LA gene fragment into pCEP4 vector, and recombinant expression vector pCLA were constructed after wards. This vector was identified by restriction endonuclease and transfected into goat fetal fibroblasts by lipofectamineTMLTX. The expression of ha-LA mRNA was detected by RT-PCR, and the expression of ha-LA protein was detected by Western-blotting. The data showed that the sequence of cloned ha-LA gene was fully correct, pCLA was constructed successfully, ha-LA mRNA was expressed in the lysate of transfected gFFCs, and ha-LA protein was expressed in the cell medium. These results suggested that the cloned ha-LA gene could correctly transcribe and translate in the goat fetal fibroblasts.