Preparation Of Porcine Rotavirus Group A Vp7 Gene Engineering Lactobacillus And Detection Of Protective Immunity

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.