Construction And Identification Of Recombinant Salmonella Choleraesuis Expressing Cap Protein Of Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2) recently emerged as an important infectious pathogen for pigs in the world.It is recognized as the major agent of post-weaning multisystemic wasting syndrome(PMWS).From the first reported in 1990s,PCV2 had widely spread in the world and maintain high infection rate and aspectrum of clinical signs,including progressive weight loss,jaundice,fever,and respiratory disease.PCV2 maintain high infection rate in herds,but most of them are inapparent infection.The virus proliferate in the immune system of piglet,and can damage immune system and induce immunosuppression.So,secondary infection often take place and cause great economic lose in the pig industry.Besides reinforcing the management of raising,keeping the environment sanitation and reducing the stress, the vaccine inoculation is the main method to prevent and control this disease. Develop safer and more effective and cheaper vaccines to prevent and control PMWS is becoming more and more important..Therefore,in the present study,for explored the new vaccine design against PCV2, we construct Salmonella choleraesuis C500 strain expressing the recombinant of Cap protein.Furthermore,the immune efficacy of the candidate vaccines was evaluated. The most research works were as following:1.Generation of monoclonal antibodies against Cap proteinThe soluble GST-Cap protein was purified with glutathione sepharose from the supernatant after the recombinant PCV2-ORF2 was induced by IPTG..Two hybridoma cell strains against Cap fusion protein were screened by indirect enzyme-linked immunosorbent assay method after fusion between Sp 2/0 myeloma cells and spleen cells BALB/c mice immunized with purified recombinant protein. Two Hybridoma cell strains were designated 3E5 and 4H8.The results of Western-blot and IFA indicated that the two monoclonal antibodies can reactione with Cap protein and PCV2 specifically.The ELISA titer of culture supernatant were 1:1024,and ascites were 1:409600 and 1:204800,Two Mabs belonged to IgG2a subclass and IgG2b subclass,and all haveκchain.This research provided a powerful tool for the research of PCV2-ORF2 gene and established the rapidly and exactly method for diagnosing PCV2. 2.Construction and identification of recombinant Salmonella choleraesuis expressing Cap protein of Porcine circovirus type 2Two pair of primes were designed according to the PCV2-ORF2 gene reported in GenBank.The whole ORF2 gene and NLS deleted ORF2 gene were amplified and subcloned into the prokaryotic expression plasmid pYA3493 containing asd gene andβ-lactamase singal sequence.The resultant plasmid pYA-ORF2,pYA-Δ41ORF2 and pYA3493 were electroporated into C500 asd mutant,resulting in the recombinant Salmonella strain C500(pYA-ORF2),C500(pYA-Δ41ORF2) and vector control C500(pYA3493).The result of SDS-PAGE indicated that the NLS deleted ORF2 gene was expressed successfully,but the whole ORF2 gene was not expressed.In addition, the result of Western-blot demonstrated that the protein expressed by NLS deleted ORF2 gene have specific reaction with anti-PCV2-ORF2 monoclonal antibodies.The C500(pYA-ORF2) and C501(pYA3493) strains remained the serum type and energy source using characterics of parent C500 strain,and the recombinant heterologous antigens can be highly expressed secretarily in C500(pYA-ORF2).Mice were orally immunized and intramuscular injection with C501(pYA-Δ41ORF2) at the dose of 5.9×10~9 CFU.All immunized groups could induce PCV2-specific humoral immune responses.The levels of IgG rose from the first week and droped from 4th week.The IgG level rose after booster immunization.