|
|
|
| Cloning and eukaryotic expression of APOBEC3G gene of AIDS patients |
| WANG Zhen-yan;J IANG Xue-yan; ZHANG Yun-zhi; LU Hong-zhou |
| Department of infectious diseases,Shanghai public health clinical center, Fudan university, Shanghai 201508, China |
|
|
|
|
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.
|
|
Received: 06 October 2008
|
|
Corresponding Authors:
LU Hong-zhou
|
|
|
|
|
|
|
