我国登革2型病毒分离株E蛋白B抗原区的表达与鉴定

目的登革2型病毒(DEN2)E蛋白B抗原区的融合表达、纯化和抗原性初步分析。方法将DEN2GZ14株E蛋白B抗原区基因连接到克隆载体pGEM-T并转染E.coliDH5α,酶切后连接至表达载体pET-32a(+)并转染E.coliBL21,IPGT诱导表达,WesternBlot和间接ELISA分析表达产物的抗原性。结果含有DEN2E蛋白B抗原区基因质粒的表达菌表达了相对分子量为31kDa的融合蛋白,WesternBlot分析表明其能与小鼠多克隆抗体发生特异性反应,间接ELISA分析其与10例登革2型病毒感染者血清发生阳性反应。结论获得的DEN2GZ14株E蛋白B抗原区的基因表达蛋白有良好的抗原性。

Expression and identification of domain B in E protein of dengue virus type 2 isolates in China

The gene encoding the domain B in E protein of dengue virus type 2 (DEN2) GZ14 strain was inserted into cloning vector pGEM-T, the recombinant plasmid was then transformed to E.coli DH5αcells. After digestion with endonucleases, it was then linked to expression vector pET-32a(+) and transfected to E.coli BL21 cells.The domain B of E gene from DEN2 was expressed with IPTG induction.Meanwhile, the antigenicity of the expressed products was tested by Western blot and indirect ELISA assay.It was demonstrated that the expression bacteria containing plasmid with gene of domain B in E protein of DNE2 could express efficiently a fusion protein with molecular weight about 31.36 kDa in E.coli BL21 cells.As indicated by Western blotting, this fusion protein could react specifically with hyperimmune mouse polyclonal antibody, and it showed positive reaction with sera of 10 patients with dengue virus type 2 infections when indirect ELISA assay was used for testing. It is evident that the good antigen icity of the product of gene encoding the domain B in E protein of dengue virus type 2 has been demonstrated.