Construction And Immunogenicity Analysis Of Recombinant Lactic Acid Bacteria Expressing Major Antigens Of Porcine Viral Diarrhea

Lactococcus lactis MG 13 63 was used as a viable recipient in the study,and its constitutive expression vector pMG36e was also used to express part of S protein of PEDV and TGEV.BALB/c female mice and 25 days old weaned piglets were orally immunized with the recombinant Lactococcus lactis respectively,and cheerful immune effects were obtained in the end.The specific IgG antibody in serum and sIgA antibody in intestinal fluid of mice were detected by double-antibody sandwich ELISA respectively.The results showed that,compared with PBS blank group and pMG36e-MG1363 group,both PEDV and TGEV of the recombinant Lactococcus lactis group were able to detect higher spectific IgG and sIgA antibody titer in serum and intestinal mucus.Four weeks after oral immunization,the spectific antibody(both serum IgG and intestinal sIgA of mice)reached the peak,and then remained at the relatively high level antibody titer for a long time.Subsequently,neutralization experiments were performed using intestinal mucosal scavengers of immunized mice,and the results showed that,compared with PBS control group and pMG36e-MG1363 group,the specific sIgA antibody was produced by intestinal mucosa of mice immunized with recombinant lactococcus lactis,which could significantly inhibit the PEDV infecting the Vero cells.Finally,the oral immunization test of weaned piglets was carried out,and specific IgG and sIgA antibodies were detected in serum and saliva of immunized piglets.It was proved that feeding recombinant lactococcus lactis could also stimulate the specific antibody in piglets’ intestinal mucosa.The expression level of IL-4 cytokines in piglets fed with lactic acid bacteria was slightly higher than that of PBS control group on 7th day and 14th day,but the difference is not significant(P>0.05).The expression level of IL-2 and IFN-y increased significantly after feeding(P<0.05),indicating that lactococcus lactis could stimulate ThO cells to differentiate into Thl cells and enhance the cellular immune response.Compared with PBS group,the expression level of IL-6 in piglets was significantly higher on 14th day,and the expression of IL-10 was not significantly different on the third day(P>0.05),but increased on 7th day and 14th day(P<0.05),and the expression level of IL-17A was increased on the 3rd and 7th day(P<0.05),and increased significantly on the 14th day(P<0.01).The results of PEDV challenge tests showed that the intestinal mucosa of duodenum,jejunum and ileum of the PBS control group’ 4 piglets have varying degrees of shedding,necrosis,accompanied by lymphatic hyperplasia and lymphoid necrosis of the submucosal layer and lamina propria.And pMG36e-MG1363 group of 3 of 4 piglets’ duodenum,jejunum and ileum intestinal mucosa also lesions.Encouragingly,only 2 of 5 piglets’ intestinal mucosal of PEDV-S318-MG1363 group lesions slightly.The above experimental results revealed that,piglets immunized with PEDV-S318-MG1363 recombinant lactococcus lactis could produce protective sIgA antibody and resist to PEDV infection to a certain extent.The protection rate up to 60%.