Preparation Of Porcine Rotavirus Group A Vp4 Genetic Engineering Lactobacillus And Detection Of Protective Immunity

Rotavirus is belong to Rotaviras of Reoviridae. RV is one of the main pathogeny of causing infant andped-age animals nonbacterial diarrhea. It is widespread epidemic in the world and caused tremendous loss,especially in the developing country. World Health organization encourage to develop RV vaccine,especially, the genetically engineering vaccine of RV. With the further investigate of molecular biology, wewere proved that VP4 was the capsid protein of RV. VP4 made virus had pathogenicity through viraladsorbing and penetration. VP4 neutralization antigen located in epitope region which could stimulate thebody to produce neutralization antibody when natural infection.In this study, Lactobacillus was selected as receptor strain to express Vp4 gene, which combinedphysiological function with specific immunity together. The aim was to lay foundation for developping thegene engineering Lactobacillus oral vaccine of Rotavirus.In this study, used SalI and KpnI as enzyme to digest the cloning plasmid pGEM-T-Vp4 andpW425et which was the prokaryotic expression shuttle vector between E.coli and Lactobacillus. The puredpositive recombinant pW42et-Vp4 was transformed into Lactobacillus acidophilus used electroporate. Thepured positive recombinant pW42et-Vp4 Lactobacillus were cultured largely, then centrifuged and addedsterilizing skimmed milk for BALB/c mouse by oral administration. After immunity, detected the level ofantibody by ELISA, the level of SIgA in intestine mucosa by SIgA kit, the change of CD3~+, CD4~+, CD8~+ Tcell subset and CD19~+ B cell subset by flow cytometry. The result indicated, the pW425et-Vp4 GeneticEngineering Lactobacillus of porcine rotavirus could enhance the immunoprotection ability of the body ofmouse.The result showed that Vp4 gene expressed in Lactobacillus and prepared the Vp4 gene engineeringLactobacillus. Furthermore Lacidlophilus may enhanced immunity of mouse, which is certificated byimmunoprotection experiment. The study is expected to lay the foundation for further studies on the geneengineering Lactobacillus oral vaccine in their prevention of Porcine Rotavirus.