Construction Of Recombinant Lactobacillus Casei Expression VP7 Of Bovine Rotavirus And Immunogenicity Analysis

Bovine rotavirus belonged to Reoviridae family, Rotavirus genus. It was the major pathogen of calf nonbacterial diarrhea. The virus usually infect 15~40 day-age calves. The clinical symptom are depressed, emesia, lose appetite, diarrhea, severe dehydration and acid poisoning. If it was accompany infected by bovine coronavirus and bacteria, the mortal rate can reach up to 80%. This disease has cost cattle producers serious loss. This disease belonged to intestinal infection and it spread through fecal-oral route, so designing a vaccine as the first line of defense etiological factor invading organism by stimulating mucosa immunity has important significance.In this study, Lactobacillus casei 393 was selected as an antigen delivery vehicle. The main protective antigen VP7 was selected as the target gene. The selective gene VP7 was directional inserted into the surface expression vector pPG-1 and secretary expression vector pPG-2, and constructed recombinant strains pPG-1-VP7/L.casei393 and pPG-2-VP7/L.casei393. Recombinant Lactobacillus casei can express and transmit exogenous antigen and itself can attach to the in intestinal tract, bear bile salt and pancreatic enzyme. So it can stimulate mucosa immunologic system of intestinal tract. The recombinant strains constructed in this study used as oral vaccine can prevent calf infecting bovine rotavirus.This study amplificated VP7 gene of Bovine rotavirus in the diarrhea stool of calf. The result of sequencing was that the gene belonged to G6 serotype and spread widely. At first, we use E.coli expression system to explore the expression condition in prokaryotic expression system and the immunity effective. The result is that 51~254 positions of VP7 protein which included major antigens can express well. Moreover, the antiserum of mouse had higher ELISA titer(1:6400) and neutralizing antibody titer(1:316).In this foundation, we constructed surface expression system pPG-1-VP7/L.casei393 and secretary expression system pPG-2-VP7/L.casei393 that can express VP7 protein of Bovine rotavirus. The recombinant strains constructed in this study were induced by lactose. The bacteria lysate and cultivate supernatant was detected by Western blot and showed that purpose protein was detected on the bacteria lysate of pPG-1-VP7/L.casei 393 and pPG-2-VP7/L.casei393. We also detected the protein on the cultivate supernatant of pPG-2-VP7/L.casei393. The indirect immunofluorescence test showed that the expressed protein was on the surface of pPG-1-VP7/L.casei 393.To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses,three groups of ten female mice were immunized respectively. Every mouse received dose of 2×109 colony-forming units (c.f.u.) of recombinant strains. The immune protocol was administered on three consecutive days at each 14 days. Collected serum, stool and vaginal lavage of mice, detected special sIgA level in the vaginal lavage, sIgA level in the stool and special IgG level in the Serum using indirect ELISA method. At the same time we detected the neutralization titer of antiserum to the virus in cellular level and estimated the immunity protection.The results of indirect ELISA showed that the mice immunized with recombinant strains could produce remarkable special sIgA level in the vaginal lavage, in the stool of mice and special IgG in the Serum of mice. The highest level of sIgA in the vaginal lavage was obtained on 14d, 37d,. and 44d after oral administration. Compared with the control group, discrepancy predominance(P<0.05). The highest level of sIgA in the stool was obtained on 23d and 44d after oral administration. Compared with the control group, discrepancy predominance(P<0.05). IgG level of the Serum of mice stepped up slowly in primary immunity and secondary immunity. The antibody level appeared apparent upgrade on the stage of the third immunization. The highest was obtained on 44d(P<0.05). The later slightly went down.The neutralizing antibody which was induced by pPG-1-VP7/L.casei393 and pPG-2-VP7/L.casei393 obtained the titer of about 1:62.9 and 1:50.6 on 48d after oral administration, respectively.The study indicated that the recombinant strains constructed in this study could induce both mucosal immune and system immune responses. And it can induce the immunity protection in group.