【作者】 韩双艳；

【导师】 郭勇； 杨慧兰； 王捷；

【作者基本信息】 华南理工大学 ， 发酵工程， 2004， 博士

【摘要】 单纯疱疹病毒Ⅱ型(Herpes Simplex Virus Ⅱ，HSV-2)主要引起生殖器粘膜感染，临床表现为生殖器疱疹(genital herpes，GH)。GH近年来发病率明显上升，成为许多国家和地区的重要性病，是我国八大性病之一。HSV-2初发感染后，可在中心神经系统潜伏，终生复发。HSV-2易经性接触传播，可形成亚临床感染、潜伏感染、复发感染，目前尚无特效药物控制其发生和复发。接种疫苗是预防和治疗HSV-2感染最有希望的方法之一。DNA疫苗可激发全面的免疫应答，是研制抗HSV-2感染疫苗的主要方向。 本研究从临床分离的野生株中克隆保护性抗原编码基因gD2(Arg92-Ala302)，构建重组真核表达质粒pVAX-gD。研究pVAX-gD在体外的表达，评价其激发动物产生免疫反应的能力及对动物的保护作用。 首先采用组织培养、透射电镜观察，分离鉴定一株造成严重复发的单纯疱疹病毒Ⅱ型野生株。根据Gene Bank提供的HSV-2糖蛋白D基因(gD2)序列设计PCR引物，特异性扩增野生株gD2全长片段，将其克隆于pUCm-T载体中。gD2基因的测序结果表明：与Gene Bank提供的HSV-1 gD DNA碱基同源性达84.6％，氨基酸同源性达82.7％；与HSV-2 gD DNA碱基同源性达98.4％，氨基酸同源性达95.7％。 BIMAS、SYFPEITHI软件分析gD2全长氨基酸序列，表明：MHC Ⅰ、Ⅱ类分子识别的抗原表位集中于野生株gD2的Arg92-Ala302之间。PCR扩增获得gD2(Arg92-Ala302)基因片段，将其与真核表达载体pVAX1在体外重组。经单双酶切鉴定，获得重组真核表达质粒pVAX-gD。 将重组真核表达质粒pVAX-gD转染哺乳动物细胞COS-7，RT-PCR检测到gD2的mRNA表达。SDS-PAGE电泳显示，pVAX-gD转染的COS-7细胞裂解液中出现一条分子量约23kD的蛋白条带，与gD2(Arg92-Ala302)理论分子量一致。免疫印迹实验表明该蛋白条带可被gD2单克隆抗体特异识别。细胞免疫组化证实gD2蛋白在细胞胞浆表达。 重组真核表达质粒pVAX-gD肌注免疫豚鼠，发现：pVAX-gD组豚鼠血清的中和指数远远大于同期pVAX组和NS组。pVAX-gD可诱导豚鼠产生高滴度gD2IgG，维持近三个月。外源特异性抗原再刺激时，迅速发生再次免疫应答。MTT法检测脾淋巴细胞增殖显示pVAX-gD具有一定的刺激脾淋巴细胞增殖能力。抗病毒攻击实验表明pVAX-gD能有效地保护豚鼠抵抗HSV-2病毒感染，延缓初发感染，减少复发，具有明显的免疫保护作用。更多还原

【Abstract】 Herpes Simplex Virus II (HSV-2) mainly causes genital tract infection at the mucosal surface and is. called genital herpes (GH) in clinic. In recent years, Occurrence of GH has increased greatly, and GH has become critical sex transimitted disease (STD) in many countries and districts. It is also one of eight STD in China. Following the initial infection, HSV-2 becomes latent in the proximal ganglion and can reactivate for life. HSV-2 can be transmitted easily through sex. It can form subclinical, latent and recurrent infection. There are no any anti-virus drugs, which can efficiently control the occurrence and recrudescence of HSV-2. Vaccine is the most promising approach to prevention and treatment against GH. DNA vaccine, which can fully induce immune response, is the one of most optimising vaccines against HSV-2.In the research, the protective antigen coding gene gD2 (Arg92-Ala302) of HSV-2 wild strain was cloned, which had been isolated from clinic specimen. The recombinant eucaryotic expression plasmid, pVAX-gD, was constructed. Expression of pVAX-gD in vitro was investigated. The ability of pVAX-gD to induce immune response in animals and the protective effect to animals were studied.A HSV-2 wild strain from clinic specimen was isolated and identified with tissue culture methods and diagnosis of transmission electron microscope, which caused serious recrudescence. PCR primers were designed according to DNA sequence of HSV-2 glycoprotein D (gD2) offered by Gene Bank. The full length gD gene of wild strain was specially amplified and cloned into pUCm-T vector. Sequencing results showed that the homology of nucleotide and amino acid between the gD of wild strain and HSV-1 gD were 84.6% and 82.7%, and that the homology of nucleotide and amino acid between the gD of wild strain and HSV-2 gD were 98.4% and 95.7%, respectively.BIMAS and SYFPEITH were used to predict antigen epitope of gD2 protein. Antigen epitopes recognized by MHC I , MHC II allele were mainly located in Arg92-Ala302 fragment of gD2 protein. gD2 (Arg92-Ala302) gene fragment was amplified by PCR and ligated into the eucaryotic expression vector pVAXl. Identified by enzyme digestion, the recombinant plasmid pVAX-gD was obtained.The recombinant plasmid pVAX-gD was transfected into mammalian cell COS-7, mRNA of gD2 gene fragment could be detected by RT-PCR in transfected cell. The results of SDS-PAGE and RT-PCR showed that there was a protein band about 23kD in更多还原