RT-PCR构建人甲胎蛋白全长基因真核表达载体

The construction of human alpha fetoprotein full length gene eukaryotic expressing vector by RT-PCR method.

目的 采用反转录PCR(RT-PCR)构建人甲胎蛋白 (Alpha-Fetoprotein, AFP)全长基因真核表达载体,为下一步应用以AFP蛋白为靶点的基因治疗作准备。方法 体外培养人肝癌细胞HepG2,Trizol裂解细胞,提取总mRNA。设计含有特定酶切位点BglⅡ,SalⅠ和真核启动元件Kozak序列的RT-PCT正反向引物,采用RT-PCR法克隆AFP全长cDNA,使用BglⅡ +SalⅠ双酶切载体pEGFP-C3载体和AFPcDNA,将AFPcDNA连接载体pEGFP-C3,构建新的真核表达载体,命名为pEGFP-AFP。结果 通过测序证明pEGFP-AFP真核表达载体含有AFP全长基因。结论:本研究成功构建人甲胎蛋白基因真核表达载体pEGFP-AFP,为进一步的分子试验和基因治疗打下了基础。

Objective: To construct eukaryotic expression vector of human alpha fetoprotein full length gene by RT-PCR method. Methods Hepatocellular carcinoma cell HepG2 was cultured in vitro. Toll mRNA were extracted after HepG2 cells were lysated by Trizol. The RT-PCR primers were designed including Kozak sequence and restriction enzyme digestion sites BglⅡ and SalⅠ. Full length AFP cDNA was cloned by RT-PCR method. The vector pEGFP-C3 and AFP cDNA were digested by BglⅡand SalⅠdouble digestion. Then AFP cDNA was ligated to vector pEGFP-C3 and the new vector was named pEGFP-AFP. Results Vector pEGFP-AFP was sequenced and the AFP gene sequence was proved correct. Conclusion Eukaryotic expression vector pEGFP-AFP was successfully constructed and it is useful to downstream study and gene therapy.