艾滋病患者APOBEC3G基因的克隆和真核表达

Cloning and eukaryotic expression of APOBEC3G gene of AIDS patients

目的建立艾滋病(AIDS)患者载脂蛋白BmRNA编辑酶催化多肽样蛋白3G(APOBEC3G)的真核表达体系。方法采用反转录-聚合酶链反应(RT-PCR)技术从AIDS患者外周血单个核细胞(PBMC)中获取APOBEC3G基因编码区,将其克隆到pMD18-T载体上,测序验证正确后再将其转接入真核表达载体pEGFP-N1中,然后将重组质粒pEGFP-N1-A3G转染HEK293T细胞,分别用RT-PCR法和蛋白印迹法(Western印迹法)验证APOBEC3G在mRNA和蛋白水平的表达。结果从AIDS患者体内克隆的APOBEC3G基因编码区长度为1154bp,测序结果与GenBank中APOBEC3G参考序列(NM021822)比对发现存在2处差异,分别位于mRNA第588位和746位碱基处。重组质粒pEGFP-N1-A3G转染HEK293T细胞,在荧光显微镜下观察到融合蛋白A3G-EGFP的表达,RT-PCR法和Westernblot法分别验证了蛋白在mRNA和蛋白水平的表达。结论成功构建了AIDS患者APOBEC3G蛋白的真核表达体系,为进一步研究APOBEC3G在HIV-1感染中的作用奠定了基础。

Objective To establish an eukaryotic expression system of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G （APOBEC3G） of AIDS patients. Methods The coding region of APOBEC3G gene was amplified by RT-PCR from the peripheral blood mononuclear cells （PBMCs） of AIDS patients, and then was cloned into pMD18-T vector. After a sequencing identification, the fragment was inserted into pEGFP-N1 to construct the recombinant eukaryotic expression vector pEGFP-N1- A3G. Transfect HEK293T cells with pEGFP-N1-A3G, and use the mothod of RT-PCR and Western blot to detect the expression of recombinant protein APOBEC3G-EGFP at mRNA and protein levels, respectively. Results The APOBEC3G gene fragment cloned into pEGFP-N1 vector consisted of 1 154 bp, of which two sites, 588 and 746 bases, were found different from APOBEC3G reference sequence （NM021822） registered in GenBank. The expression of recombinant protein APOBEC3G-EGFP was observed in HEK293T cells by fluorescence microscope, detected by RT-PCR for mRNA level, and identified by Western blot for protein level. Conclusion Eukaryotic expression system for APOBEC3G of AIDS patients was successfully constructed, which lays a foundation for the study of APOBEC3G＇s role in HIV-1 infection.