不同启动子对重组痘苗病毒表达狂犬病病毒糖蛋白基因的影响

Effects of Different Promoters on Expression of Rabies Virus Glycoprotein Gene in Recombinant Vaccinia Virus

以血凝素（ＨＡ）基因为侧翼，将狂犬病病毒糖蛋白基因ｃＤＮＡ置于不同类型的痘苗病毒复合启动子下游，构建了４种表达质粒。重组表达质粒转染已感染ＷＲ株痘苗病毒的ＢＨＫ２１细胞，并通过鸡红细胞吸附试验，筛选、纯化出与表达质粒对应的４组ＨＡ－重组痘苗病毒：ｖＳＦＪ１－１０ＲＶｇｐ，ｖＳＦＪ２－１６ＲＶｇｐ，ｖＲＪ１－１０ＲＶｇｐ，ｖＲＪ２－１０ＲＶｇｐ。经间接免疫荧光试验（ＩＦＡ）鉴定，从４组重组病毒中每组各鉴定出１株阳性重组病毒。Ｗｅｓｔｅｒｎｂｌｏｔ检测显示，重组病毒感染的ＢＨＫ２１细胞裂解物上清中有１种蛋白与抗ＲＶＧＰ的ＭｃＡｂ发生特异反应，分子量为６９０００。在重组病毒感染早期，ＲＶＧＰ的表达量以ｖＳＦＪ１－１０ＲＶｇｐ最高；而在感染晚期，则以ｖＳＦＪ２－１６ＲＶｇｐ最高。４株重组病毒免疫小鼠，均能够不同程度地诱导小鼠产生抗ＲＶＧＰ特异性抗体。

With flank sequence of HA gene of vaccinia virus, four eucaryotic expression plasmids containing cDNA of rabies virus glycoprotein gene under the control of different hybrid promoters of pox virus were constructed and transfected into BHK 21 cells infected with wild type vaccinia virus. Four RVgp recombinant vaccinia viruses (rVV): vSFJ1 10RVgp, vSFJ2 16RVgp, vRJ1 10RVgp and vRJ2 10RVgp were screened and cloned by hemadsorption in BHK 21 cells, and further characterized with indirect immunofluorescence assay (IFA). IFA and Western blot demonstrated that the RVGP was fully expressed by all rVVs in BHK 21 cells. Western blot showed that the protein in supernatants of infected BHK 21 cells' lysate was able to react specifically with anti RVGP McAb, with it's molecular weight being 69 000. RVGP induced by vSFJ1 10RVgp accounted for the most quantity in the early stage of rVV infection and was as 2 fold as that of the others. In the late stage, vSFJ2 16RVgp was able to product the most output as 2 8 folds as that of the other rVVs. The results also indicated that the RVgp recombinant VVs could induce different titers of specific antibody in mice.