HIV-2gag基因在重组痘苗病毒中的表达

Expression of HIV2 Gag Gene in Recombinant Vaccinia Virus

以痘苗病毒ＨＡ基因为侧翼，将ＨＩＶ－２ｇａｇ基因部分序列（简称Ｇ１）和全部序列（简称Ｇ２）分别插入牛痘病毒Ａ型包涵体（ＡＴＩ）和串联１０个（与ＨＡ基因反向）或１６个（与ＨＡ基因同向）痘苗病毒Ｐ７．５组成的复合型启动子下游，构建了４个重组痘苗病毒表达载体质粒。经脂质体转染、鸡红细胞吸附试验筛选和免疫荧光鉴定，获得４株能稳定表达目的蛋白的重组痘苗病毒。实验结果表明，以ＨＡ基因为侧翼构建重组病毒的重组率约为０．１％，阳性重组率为２５％～１００％。实验中还发现，以ＨＡ基因为侧翼的重组痘苗病毒能够引起细胞融合，其可作为筛选重组痘苗病毒的另一种标志。Ｗｅｓｔｅｒｎｂｌｏｔ结果表明，表达的Ｇａｇ蛋白能被ＨＩＶ－２血清中的特异性抗体所识别，并能诱导小鼠产生抗ＨＩＶ－２Ｇａｇ抗体。

Four eucaryotic expression plasmids containing cDNA of HIV2 gag gene under the control of different hybrid promoters of pox virus and 10 or 16 tandemly repeated mutant early P7.5 were constructed, and transfected into BHK21 cells which were infected by wild type vaccinia virus. The recombinant rate of 0.1%, positive recombinant rate of 25%100% and the syncytium formation to great extent with flank sequence of HA gene of vaccinia virus were observed. Indirect immunofluorescence assay (IFA) and Western blot showed that all the recombinant vaccinia viruses expressed the Gag protein which can specifically response to HIV2 positive serum, and induce HIV2 Gag specific antibody in mice.