轮状病毒组特异性抗原VP6在重组腺病毒中的表达及其免疫学效果的研究

目的 构建可表达A组轮状病毒组特异性抗原VP6的非复制型重组腺病毒载体，并对其生物学和免疫学性质进行研究。方法 将人轮状病毒VP6基因插入腺病毒载体pShuttle-CMV中，通过细胞内同源重组方法获得重组腺病毒DNA，将其转染293细胞获得重组腺病毒。用聚合酶链反应（PCR）及Southern blot方法，检测目的基因在重组腺病毒中的整合，用Western blot检测VP6的表达。通过灌胃和滴鼻两种途径对小鼠进行免疫，并对免疫后小鼠体液和粘膜免疫进行分析。结果 得到了重组腺病毒rvAd-VP6,VP6基因整合于腺病毒基因且中，在293细胞中可稳定表达。2次免疫后灌胃和滴鼻两组小鼠均产生明显免疫应答，血清IgG抗体滴度分别为1：1000和1：10000－1：100000。除了血清IgG外，小鼠还产生了较强的针对轮状病毒的血清IgA，滴度为1：10－1：100。滴鼻组在肺灌洗液和肠匀浆液中均可检测到分泌型IgA(sIgA)，灌胃组仅在肠道检测到sIgA。滴鼻组的免疫学效果明显优于灌胃组。结论 人轮状病毒VP6基因重组腺病毒载体的成功构建及所取得的良好免疫学效果，为我国具有自主知识产权的新型轮状病毒基因工程疫苗的研制奠定了基础。

Expression of the main structural antigen VP6 of human rotavirus by recombinant adenovirus and immune responses induced in vivo

BACKGROUND: Constructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo. METHODS: The cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA. Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot. BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus. RESULTS: Recombinant adenovirus named rvAd-VP6 was obtained. The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably. The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively. In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100.Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group. The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation. CONCLUSIONS: The recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.