临床分离2型登革病毒突变株E蛋白N端158氨基酸基序免疫原性初探

目的:Ⅱ型登革病毒临床分离突变株(B株)E基因区部分序列的原核表达载体构建,蛋白表达、复性和纯化,及其免疫原性的初探。方法:将B株E基因1～476bp序列克隆入原核表达载体pET28a(+),经酶切、测序鉴定正确后转入表达菌,IPTG诱导后将包涵体变性,复性,纯化后的蛋白免疫C57BL/6和BALB/c小鼠制备多克隆抗体,并通过Western blot和ELISA进行抗体鉴定。结果:成功构建了pET28a(+)-B-E原核表达载体,在表达菌中以包涵体形式稳定高表达,分子量为20kD。利用蛋白的His标签进行Western blot鉴定确定该蛋白为重组B-E蛋白,得到的可溶蛋白纯度大于90%。B-E蛋白免疫BALB/c和C57BL/6小鼠均可以得到有效的多克隆抗体。结论:B-E蛋白对BALB/c和C57BL/6小鼠具有免疫原性,其中ELISA鉴定免疫C57BL/6小鼠抗体的滴度为1∶12800,Western blot鉴定免疫BALB/c小鼠抗体滴度为1∶500。

Immunogenicity of N-terminal 158 amino acid motif of E-protein of a newly purified mutant dengue virus from a Chinese patient

Objective:The envelope protein Domain I(DI) of type II mutated dengue virus from a DHF patient (B-E) was expressed,refolded and purified.Its immunogenicity was also identified.Methods:The 1-476 bp of B-E was cloned into pET28a(+),and it was transformed into E.coli after digestion and sequencing.The inclusion body was denatured and refolded after inducing with IPTG.Polyclone antibody was obtained by immunizing C57BL/6 and BALB/c mice with purified B-E,and identified with Western blot and ELISA.Results:The cD...