表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。

Construction of recombinant vaccinia virus expressing HSV-2 gD gene as live recombinant vaccine strain

We had reported that the recombinant vaccinia virus expressing glycoprotein D of herpes simplex virus type 2 (HSV-2 gD) protected mice against lethal HSV-2 challenge. Following the succeed in animal model, we continue the research to construct the recombinant vaccinia virus expressing HSV-2 gD gene as live recombinant vaccine strain in strict accordance with the guideline for human vaccine research. A PCR-modified HSV-2 gD gene was inserted into the plasmid pJSB1175, under the control of P7.5K early/late promoter of vaccinia virus. The recombinant plasmid was used, with lipofectin reagent, to transfect 2BS cells which had been infected by wild type of TK vaccinia virus (Tian Tan 761 strain). The recombinant vaccinia virus harboring HSV-2 gD gene was selected out by using in situ hybridization employing 32 P-labelled HSV-2 gD fragment as probe, together with three cycles of plaque purification. Dot and Southern blot confirmed HSV-2 gD gene had been integrated into the TK region of vaccinia virus genome, as expected. Indirect immunofluorescent assay using anti-HSV-2 gD monoclonal antibody showed HSV-2 gD was expressed effectively in the recombinant virus infected cells.