Structure The Restructuring Of IBDV VP2-Live Carrier Vaccine Strains Of Lactic Acid Bacteria And Test The Invasion Ability

Lactic acid bacteria(LAB) is gastrointestinal probiotics of the human and animal, it is generally regarded as safe(GRAS). LAB enable immunization via the mucosal route, which increases effectiveness against pathogens that use the mucosa as the major route of entry into the human and animal, which made it have vital significance and application potential.Aim: In this study Lactococcus NZ3900 was used as genetic engineering tools to clone the antigen protein VP2 of infectious bursal disease virus and Salmonella invasion protein RCK to structured three recombinant Lactococcus, L.Lactis-VP2, L.Lactis-RCK and L.Lactis-VP2-RCK. In vitro experiment, the function of RCK protein and VP2 protein expressed by recombinant Lactococcus, was identified. In vivo experiment, recombinant Lactococcus L.Lactis-VP2.Method: In this study, the VP2 and RCK gene were obtained by polymerase chain reaction(PCR), and inserted into the food-grade vector pNZ8149 plasmid. After the verificated, it was transformed into NZ3900 by electroporation. Nisin was used as the revulsant to express the recombinant protein, SDS-PAGE and Western Blot results showed that the recombinant protein was expressed correctly and have the high stability, further more the location of the recombinant protein was also detected. To improve the efficiency of the recombinant protein, the codon of exogenous gene was optimized and the expression condition was optimized through the orthogonal experiment, the orthogonal experiment result showed that at the time OD600=0.5 begin to expression for 5 hours with concentration of nisin 2 ng/ml obtained the best content of recombinant protein as 38.0 ug/ml detected by BCA kit and thin layer of gel scan. In vitro gentamycin survival assay and Confocal laser scanning microscopy observation in MA104 cells and HT29 cell showed that the recombinant Lactococcus can obtain the ability of invasive by expressing RCK protein.Result:Base on the research above, we structure another recombinant Lactococcus which can express the fusion protein VP2-RCK, In vitro gentamycin survival assay, Confocal laser scanning microscopy and Scanning electron microscope observation results all suggest that the fusion by RCK and VP2 protein was not affect the function, it both have the immunogenicity of VP2 protein and the invasive ability of RCK protein.