Construction Of Spore Surface Display Porcine Rotavirus VP8 Portein And Detection Of Immunity Effect

Porcine rotavirus causes severe diarrhea as the main symptom of infectious diseases,results in huge economic loss in pig industry of the world.Pigs of all ages can be infected with rotavirus,and nursing piglets have more severe symptoms,weaned piglets characteristically only minimal to moderate or no clinical manifestations but can continue to shed the virus to environment.The virus is transmitted by the fecal-oral route and survive in the environment for a long time.Therefore,swine herds once infected with rotavirus are difficult to eliminate.There has been no effective treatment for rotavirus infection.And we can only take complementary symptomatic treatment to prevent dehydration and acidosis and prevent secondary infection.Vaccination is considered to be an effective measure to control these infection.In this study,we constructed a recombination B.subtilis strain with spore surface display heterologous antigen protein VP8 of porcine rotavirus and evaluated the immunogenicity of recombination strain.The aim is to develop a substitute porcine rotavirus mucosal subunit vaccine candidate against rotavirus infection worldwide.The studies and results are as follows:1.Design and biosynthesize a pair of primer based on the sequence of porcine rotavirus VP4 in NCBI/Gen Bank.The VP4 gene is amplified and insert it into p MD-19T simple vector.And the nucleotide sequence and evolution trees showed that the VP4genotypes was P[7].Design and biosythsize a pair based on the sequence of VP4.And the cloned PCR product VP8 gene was inserted into p MD-19T.2.An E.coli-B.subtilis shuttle vector(p DG364-Cot B-VP8)was constructed for surface display VP8 on the spore of B.subtilis 168 to obtain B.subtilis BV.The integrity and fidelity integrity of fragment were confirmed by agarose gels electrophoresis analysis of double restriction enzyme digested and DNA sequencing.Plasmids p DG364-Cot B-VP8was linearized by digestion with Xba?and used to transform competent cells of B.subtilis 168.All Cm^rclones were tested the amylase activity.And chromosomal DNA extracted from Cm^rclones and amylase inactivated strain was used to PCR identification with several different pairs of primers.Immunofluorescence was used to determine VP8protein surface expression on the spore.Spore coat proteins extraction from the purified spores B.subtilis BV were tested by Western Blot.3.Mice were randomly divided into five groups(25 mice in each group).The control group(A)was fed with a basal diet.Two groups were fed a diet with 5.0×10⁶CFU/g spores B.subtilis 168(B)and B.subtilis BV(D)respectively.The group C(B.subtilis 168)and E(B.subtilis BV)were orally immunized by intragastric gavage containing 2.0×10¹⁰CFU/g spores in a volume of 0.15 m L on days1,2,3,14,15,16,28,29 and 30.Serum and fecal sample were collected on days 0,14,21,28 and 35 for specific anti-VP8 antibody Ig G and SIg A by ELISA.The specific Ig A levels in feces of mice with two administered methods induced by B.subtilis BV spore increased from 14 days to 35 days than B.subtilis 168(P<0.01),and the group D is higher than group E on days 21 and 35(P<0.05).The serum Ig G antibody elicited by B.subtilis BV treated with two administered methods were similar to each other from 14 days to 35 days but higher than B.subtilis 168(P<0.01).4.The weight of mice,spleen,thymus were measured and cecum contents were collected for the plate count and PCR-DGGE on days 35.The results showed that the weight increase of B,C,D,E groups were highter than group A and the differences are remarkable(P?0.05).The quantity of E.coli in the cecum contents of Group A is higher than other groups(P?0.05),and the Lactobacillus and Bifidobacterium are lower,but there is no significant differences(P>0.05).Spenic index of B,C,D,E groups increased(P?0.05)than group A,and thymic index was no significant difference(P>0.05).