DNA疫苗pRSC-gD.gC-IL-21的构建及其预防HSK的动物实验初步研究  

Construction of DNA vaccine pRSC-gD. gC-IL-21 and preliminary research on its immunoprotection against HSK in mice model

作　　者：董莉莉[1,2] 胡凯[1,3] 汤明霞[1] 

机构地区：[1]东南大学医学院眼科学系,江苏南京210009 [2]江苏省泰州市人民医院眼科,江苏泰州225300 [3]南京大学医学院附属鼓楼医院眼科南京宁益眼科中心,江苏南京210008

出　　处：《东南大学学报（医学版）》2016年第6期932-938,共7页Journal of Southeast University(Medical Science Edition)

摘　　要：目的:构建DNA疫苗pRSC-gD.gC-IL-21,探讨其是否能诱导动物免疫应答及抵抗眼角膜单纯疱疹病毒Ⅰ型(HSV-1)的感染。方法:提取HSV-1基因组DNA,PCR扩增获得gC基因。以pRSC-gD-IL-21质粒为模板(本实验室2011年构建),构建重组质粒pRSC-gD.gC-IL-21,用纳米材料壳聚糖(chitosan)包裹,采用黏膜接种的方式分别以pRSC-gD.gC-IL-21+chitosan、pRSC-gD-IL-21+chitosan、pRSC+chitosan及chitosan免疫小鼠3次,间隔2周,末次免疫2周后检测各项免疫学指标,同时评价小鼠对病毒攻击角膜的免疫保护作用。结果:经测序、酶切及Westernblot鉴定,重组质粒pRSC-gD.gC-IL-21构建成功,chitosan包裹率高。与对照鼠相比,该疫苗在鼠体内产生了更强的特异性中和抗体、细胞毒性T淋巴细胞(CTL)及自然杀伤细胞(NK)杀伤活性增强、泪液中特异性sIgA水平增高,同时使小鼠产生了较强的针对眼角膜HSV-1感染的免疫保护作用。结论:DNA疫苗pRSC-gD.gC-IL-21构建成功,能诱导小鼠产生较强的免疫应答及针对HSV-1眼部感染的免疫保护作用,能够预防单纯疱疹病毒性角膜炎(HSK)的发生、发展。Objective： To construct DNA vaccine pRSC-gD. gC-IL-21 and to investigate its ability to induce immune response and resist the herpes simplex virus type 1（ HSV-1） infection of cornea in mice. Methods： HSV-1 genomic DNA was extracted and the gene of gC was amplified by PCR. As a template with pRSC-gD-IL-21plasmid（ in 2011, our laboratory has been building successful）, DNA vaccine pRSC-gD. gC-IL-21 was constructed. After being identifed by molecular methods,we packaged them with nanometer materials chitosan.With the method of mucosal immunization,the vaccine pRSC-gD. gC-IL-21 ＋ chitosan,the pRSC-gD-IL-21 ＋chitosan,the blank pRSC ＋ chitosan and the chitosan were respectively inoculated into BALB / c mice for 3 times with 2 weeks interval. A number of immunological indexes were detected 2 weeks after the last immunization. At the same time,the immunoprotective against HSV-1 challenging on cornea in mice was also evaluated. Results： By DNA sequencing,the restricted endonuclease analysis,and Western blot expression,the construction of DNA vaccine pRSC-gD. gC-IL-21 was proved to be constructed successful,and chitosan package rate was high. Compared with the control mice,the DNA vaccine induced the mice to generate higher levels of antibody,enhanced the splenic cell proliferation response as well as the activities of cytotoxic T lymphocyte（ CTL） cell and natural killer（ NK） cell,and increased the specificity sI gA levels in tears. Meanwhile,the pRSC-gD. gC-IL-21 ＋ chitosan could elicit a stronger immunoprotective effect against the corneal infection of HSV-1 in mice. Conclusion： The construction of DNA vaccine pRSC-gD. gC-IL-21 is successful. The DNA vaccine pRSC-gD. gC-IL-21 may induce an effective immune response in immunized mice. More importantly,the efficacy against the HSV-1 challenge in mouse cornea is enhanced markedly. It can prevent the occurrence and development of the herpes simplex keratitis（ HSK）.

关 键 词：单纯疱疹病毒1型 DNA疫苗 糖蛋白C 壳聚糖 单纯疱疹病毒性角膜炎 小鼠 

分 类 号：R392.11[医药卫生—免疫学]