我国HIV-1 B'/C重组流行株Tat蛋白的表达、纯化及功能分析

High Expression,Purification and Functional Analysis of the Tat Protein of HIV-1 B'/C Recombinant Strains in China

根据全国HIV分子流行病学研究发现,B'和C亚型HIV-1在我国发生了重组,并以优势株形式在我国广泛流行。为了探讨这种HIV-1B'/C重组毒株tat基因的变异与其表型之间的关系,利用pET表达系统在大肠杆菌中高效表达了三种不同基因变异类型的Tat蛋白,重组蛋白占菌体总蛋白的26%,Westernblot显示较好的反应原性,并通过金属鏊合层析纯化了目的蛋白。荧光素酶活性检测表明:体外表达的Tat蛋白具有明显的生物学活性,可以反式激活HIVLTR引导的报告基因的表达;三种Tat蛋白在激活活性上的差异与流行现场检测的病毒载量的高低存在明显的对应关系,说明tat基因的变异可以引起病毒生物学特性的改变,进而影响病毒的流行特征。此结果为进一步研究我国HIV重组毒株的基因变异特征及变异规律奠定了基础。

In the first survey on national HIV molecular epidemiology,we found that the HIV-1 B'/C recombinant strains were prevalent in the northwest of ChinaTo explore the relationship between the genotype and phenotype of mutatied tat genes,three tat genes of the HIV-1 B'/C recombinant strain in China with different mutations were cloned and highly expressed in Escherichia coli using the prokaryotic expression vector pET 30aThe average ratio of expression of each recombinant Tat protein to total bacteria proteins was 26%Western blot showed good reactivity to monoclonal antitat antibodyTat proteins were purified by nickelchelating chromatographyLuciferase assays indicated that the Tat proteins expressed in vitro had biological activity,and could transactivate the downstream reporter gene of HIV-1 LTRThe difference in transactivation was in direct proportion to the viral load of the HIVinfected individualsThe above results show that tat gene mutation can change its phenotype,and further impact viral epidemiology This study sheds light on patterns of gene mutations and characteristics of the HIV-1 B'/C recombinant strains in China