【作者】 胡静涛；

【导师】 王春凤；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 轮状病毒(Rotavirus，RV)是引起婴幼儿和各种幼龄动物非细菌性腹泻的主要病原之一。由于目前尚无治疗轮状病毒的特效药物，因此预防显得尤为重要。为此，世界卫生组织多次呼吁各国科学家研制开发有效的轮状病毒疫苗，并将其列为最优先发展的疫苗项目之一。随着分子生物学研究的不断深入，现已明确VP7是RV的主要外壳蛋白和保护性抗原之一，在疫苗的研制中具有重要的意义。本实验选择乳酸菌作为受体菌株表达外源基因Vp7，即可将乳酸菌的益生功能和外源基因的特异性免疫相结合，从而研制猪轮状病毒Vp7基因工程乳酸菌。目的在于为开发研制预防猪轮状病毒病的基因工程乳酸菌口服疫苗奠定实践基础。在本研究中，将猪A组轮状病毒主要保护性抗原Vp7克隆至可在大肠杆菌和乳酸菌中穿梭表达的具有红霉素和胸苷酸基因的双标记表达载体。经过优化电转化条件，将重组质粒pW425et-Vp7转化到乳酸菌受体菌中，通过乳酸菌质粒的提取，酶切分析和PCR方法鉴定阳性克隆质粒。将筛选好的阳性克隆进行SDS-PAGE及Western bloting检测，结果表明外源基因Vp7在乳酸菌中获得了表达。将可以表达的重组pW425et-Vp7基因工程乳酸菌给4～5周龄的BALB／c小鼠连续灌服，免疫期30天。分别在第5、10、15、20、25、30d灌服1h后，摘除眼球采集血清及肠道内容物，采用间接ELISA方法检测血清抗体和SIgA的水平。另外，免疫后每隔10天，采用流式细胞仪检测CD3~+、CD4~+、CD8~+T细胞和CD19~+等B细胞的相对变化情况，最后用SPSS11.0统计软件对以上所得数据进行分析比较。结果表明，猪A组轮状病毒Vp7基因在乳酸菌中获得了表达，并制备了猪轮状病毒基因工程乳酸菌。将猪轮状病毒Vp7基因工程乳酸菌给小鼠免疫后，具有较好的免疫效果。从而为进一步研究轮状病毒基因工程乳酸菌口服疫苗奠定基础。更多还原

【Abstract】 Rotavirus is one of the main pathogeny of causing infant and virous kinds ped-age animalsnonbacterial diarrhea. At present, dueing to the patent medicine is not manufactured, so the prevention ismore important. WHO appealed scientists in countries to develop effective Rotavirus vaccine many times,and took it as one of the most priority developed proceedings. Along with lucubration of molecular biology,main coat protein VP7 is not only the base of RV serotype typing, but also the main protective antigen.In this study, L.acidlophilus was selected as receptor strain to express Vp7 gene, which combinedphysiological function with specific immunity together. The aim was to lay foundation for developping thegene engineering L.acidlophilus oral vaccine of Rotavirus.In this study, Vp7 gene of Rotavirus was cloned into expressing vector with Erythromycin and ThyAdouble labellings, which can shuttle and express in E.coli and L.acidlophilus. After optimizing thecondition of electroporation, the recombinant plasmid pW425et-Vp7 was transformed into the competenceof L.acidlophilus. And the positive clone was detected by plasmid extraction, enzyme digestion and PCRidentification. Vp7 gene was confirmed to express in the L.acidlophilus by SDS-PAGE and Westernblotting. At last gene engineering L.acidlophilus with plasmid pW425et-Vp7 was continuously drenched tothe 4～5weeks BALB/c mouse, The stage of immunization was 30 days. The whole blood Serum wascollected by extirpating eyeballs. The mucosa and content of intestinal tract were also gathered, after 510、15、20、25、30d of immunization 1h. The above-mentioned sample was detected by indirect ELISA forthe level of the antibody of blood and SIgA. At the same time, the relative quantity of CD3~+,CD4~+,CD8~+and CD19~+ in the spleen was detected by flow cytometer(FCM) at intervals by 10 day of immunization.The data was analyzed by SPSS11.0 statistic software.The result showed that Vp7 gene expressed in Lactobacillus and prepared the Vp7 gene engineeringLactobacillus.Furthermore L.acidlophilus may enhanced immunity of mouse, which is certificated byimmunoprotection experiment. The study is expected to lay the foundation for further studies on the geneengineering L.acidlophilus oral vaccine in their prevention of Porcine Rotavirus.更多还原