Preliminary Study On VP6 Subunit Vaccine Of Group A Bovine Rotavirus

Group A bovine rotavirus(Group A Bovine Rotavirus,BRVA)is one of the most important pathogens of diarrhea in calves.BRVA mainly infects calves less than 7 days old,causing severe diarrhea and even dehydration and death of calves,resulting in serious economic losses to the cattle industry.BRVA VP6 protein is a highly conserved viral capsid protein,which can induce broad-spectrum neutralization response and is a candidate antigen for subunit vaccine.(LTB),the heat-labile enterotoxin B subunit of Escherichia coli,specifically binds to mucosal epithelial cells and can be used as a mucosal immune adjuvant.The purpose of this study is to develop BRVA VP6+LTB subunit vaccine,establish an indirect ELISA method to detect mouse anti-BRVA SIgA antibody,and evaluate the immune effect of the vaccine in mouse model.The results are as follows:1.LTB protein was successfully expressed and purifiedIn this study,the recombinant LTB protein was prepared by using E.coli as expression system.The prokaryotic expression plasmid pET28-LTB,was transformed into E.coli BL21(DE3),and the expression was induced by IPTG.The purified product was purified by Ni2+-NTA purification column,and the purified product was dialyzed and renatured.The binding activity of the renatured product to GM1 was identified by ELISA.The results showed that the prokaryotic expression vector was successfully constructed and the size of the recombinant protein was 14 KDa.The recombinant bacteria were induced to express at 37℃ for 6 h or overnight at 20℃.The recombinant protein was expressed in the form of inclusion body;the purity of the purified and renatured recombinant protein could reach 90%.The recombinant protein reacted with rabbit anti-LT serum and had good reactivity;it could specifically bind to cell surface GM1 and had adjuvant activity.This study lays a foundation for the preparation of mucosal adjuvants for VP6 subunit vaccine.2.Establishment of indirect ELISA method for mouse anti-group A bovine rotavirus SIgA antibodyThe BRVA VP6 protein prepared in the laboratory was used as the coating antigen and the HRP labeled sheep monoclonal antibody against mouse SIgA secretory component(J chain)was used as the enzyme labelled second antibody.After the optimization of reaction conditions and system,an indirect ELISA method was established to detect BRVA specific SIgA antibody in mouse small intestinal mucosa and vaginal mucosa.The results showed that the best coating concentration of VP6 protein was 2.5 μg/mL.The best dilution ratio of small intestinal mucosa antibody was 1:20,and the positive result was determined as OD450nm≥0.245.The best dilution of vaginal mucosa antibody is 1:5,and the positive result was OD450nm≥0.189.This method did not cross-react with intestinal mucosal antibodies induced by bovine coronavirus and bovine viral diarrhea virus infection.The coefficient of variation of the repetitive test was 0.186%-5.85%,and the results of positive intestinal mucosal antibodies diluted 80 times were still positive.This method has good specificity,repeatability and sensitivity.It provides a technical means for the follow-up detection of the level of mucosal immune antibodies in mice induced by VP6 subunit vaccine.3.BRVA VP6 LTB subunit vaccine stimulates systemic and mucosal immune response in mice and induce protective effects in miceIn order to evaluate the immune effect of BRVA VP6+LTB subunit vaccine on mice,BALB/c adult female mice were divided into three groups,which were immunized with different doses of subunit vaccine,30 μL/mouse(antigen content was 10 μg VP6+10 μg LTB,20 μg VP6+10 μg LTB,30 μg VP6+10 μg LTB respectively).There was also a group of subcutaneously injected immune subunit vaccine,30 μL/mouse(antigen content is 30 μg VP6+10 μg LTB),with 5 mice in each group,which wre immunized once every 2 weeks with the same dose and way.On the 14th day after the second immunization,the levels of serum IgG,SIgA antibody in intestinal mucosa and SIgA antibody in vaginal mucosa were determined by indirect ELISA.Results Intranasal immunization with 30 μg VP6+10 μg LTB antigen could not only induce IgG antibody in serum(OD450nm=1.009±0.103),but also induce specific SIgA in intestinal mucosa(OD450nm=1.207±0.219)and vaginal mucosa(OD450nm=0.865±0.108)The prepared subunit vaccine can effectively stimulate the systemic and mucosal immune responses of mice by intranasal immunization.In order to further study the protective effect of VP6 subunit vaccine on suckling mice,the intranasal immunized female mice(30 μg VP6+10 μg LTB)were mated after the second immunization,and the challenge test was carried out on the suckling mice.The diarrhea of suckling mice was observed within 96 h after challenge and the virus load in feces was detected.The results showed that severe diarrhea occurred in all the suckling mice in the control group within 48-96 h after challenge,and no severe diarrhea occurred in the suckling mice with maternal antibodies during the 96 h observation period,and the virus load in the feces of the two groups increased to the highest at 72 h.The virus load in the maternal antibody group was significantly lower than that in the control group by 5 times.(P<0.01).The results showed that the prepared VP6 subunit vaccine could stimulate maternal mice to produce high levels of maternal antibodies,inhibit virus replication in the intestinal tract,alleviate clinical symptoms of severe diarrhea,and protect suckling mice by passive immunization.It laid a foundation for further study on the immune efficacy of VP6 subunit vaccine in cattle.4.Correlation and crossover neutralization activity of mucosal antibodies induced by BRVA VP6 subunit vaccine.The correlation between the OD450nm value of vaginal mucosal specific SIgA antibody and the OD450nm value of intestinal mucosal specific SIgA antibody was analyzed,and the neutralizing activity of vaginal mucosal antibody against bovine rotavirus(SGⅡ,G6P[1])and yak rotavirus(SGⅡ,G8P[1])was determined by microneutralization test.The results showed that the correlation coefficient between them was R2=0.8889.The vaginal mucosal antibody of mice immunized with vaccine could inhibit the infection of two strains of BRVA to MA104 cells(neutralizing titer were 1:820 and 1:512).It is suggested that the detection of specific vaginal mucosal SIgA antibody can be used as an alternative index to reflect the local mucosal immunity of small intestine,and the mucosal antibody induced by subunit vaccine has cross-neutralizing effect on different genotypes of BRVA.