Cloning And Expression Of The Human Apobec3g Gene And The Expression Of Hiv-1 Infection By Apobec3g/b/f

Objective To establish an eukaryotic expression system of human apolipoprotein B mRNA-editing enzyme,catalytic polypeptide-like 3G(APOBEC3G, hA3G),which had been identified an innate anti-HIV-1 factor in vitro.Methods The coding region of hA3G gene was amplified by RT-PCR from peripheral blood mononuclear cells(PBMC) isolated from an HIV-infected patient,then cloned it into a T vector.After a sequencing identification,the coding region of hA3G gene was inserted into eukaryotic expression vector pEGFP-N1.Then transfect HEK293T cells with recombinant pEGFP-N1-A3G,and detect the expression of recombinant protein hA3G-EGFP by RT-PCR and Western Blot for mRNA and protein levels respectively. Results The hA3G gene fragment cloned into pEGFP-N1 vector consisted of 1 154bp, of which two sites,mRNA 588 and 746 bases,were found different from hA3G reference sequence(NM021822) registered in GenBank.The expression of recombinant protein hA3G-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 An eukaryotic expression system for hA3G was successfully constructed,which lay a foundation for the study of its role in HIV-1 infection. Objective Human cytosine deaminase family APOBEC3(hA3) was found to have antiretroviral activity in vitro.Of its members,hA3G and hA3F were identified potent inhibitors of vif-deficient HIV-1,and another member hA3B was able to suppress the infectivity of both vif-deficient and wild-type HIV-1.Our study is to quantitative investigate the expression levels of hA3G,hA3B and hA3F in peripheral blood mononuclear cell(PBMC) of HIV-1-infected subjects,then analyze there correlation with CD4~+T cell counts,expecting to demonstrate their potential roles in delaying HIV-1 disease progression.Methods Collect peripheral blood samples from 21 HIV-1-infected subjects not taking antiretroviral therapy(ART),21 HIV-1-infected subjects receiving ART,and 10 HIV-1-uninfected controls,then separate PBMC by Ficoll density gradient centrifugation,followed by total RNA extraction with TRIzol and cDNA synthesis,hA3G/hA3B/hA3F mRNA levels were determined by real-time fluorescent quantitative PCR.Flow cytometry was used to detect CD4~+T cell counts.Results There were no correlations between hA3G/hA3B/hA3F mRNA levels and CD4 counts in either ART+ or ART-HIV-1-infected subjects,hA3G mRNA level in HIV-1-infected subjects was lower than that in HIV-1-uninfected controls(P＜0.05),but no statistical difference was found between ART+ and ART-groups(P＞0.05).However,significant differences were found for hA3B/hA3F mRNA levels between the three groups(P＜0.05):ART-HIV -1-infected subjects＜ART+HIV-1-infected subjects＜HIV-1-negative controls. hA3G/hA3B/hA3F mRNA levels were positively correlated with one another in ART+ HIV-1-infected and HIV-1-uninfected subjects,but not in HIV-1-infected subjects not taking ART.Conclusion hA3G/hA3B/hA3F expression levels do not directly relate with HIV-1 disease progression.Their expression levels tend to decrease after HIV-1 infection, but ART may elevate hA3B/hA3F mRNA levels,but not for hA3G.The expression of hA3G,hA3B and hA3F may be suppressed at different degrees when HIV-1 replicates at different levels.