摘要
目的 建立艾滋病( AIDS) 患者载脂蛋白B mRNA 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1 154 bp, 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Western blot 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。
Abstract
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.
关键词
真核表达 /
艾滋病
Key words
APOBEC3G /
Eukaryotic expression /
Acquired immunodeficiency syndrome
王珍燕1,3 ; 江雪艳1 ; 张云智1 ; 卢洪洲1,2,3.
艾滋病患者APOBEC3G 基因的克隆和真核表达[J]. 微生物与感染. 2008, 4(4): 219-223
WANG Zhen-yan;J IANG Xue-yan; ZHANG Yun-zhi; LU Hong-zhou.
Cloning and eukaryotic expression of APOBEC3G gene of AIDS patients[J]. Journal of Microbes and Infections. 2008, 4(4): 219-223
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
PDF(1196 KB)
