| Porcine circovirus disease, Japanese encephalitis and porcine parvovirusinfection can cause reproductive failure of pigs. Theses three diseases have broughthuge economic losses to pig industry because of performance for mixed infection orsecondary infection. Vaccination is an effective method to prevent and control thethree major diseases, but there is only single vaccine commercially availablecurrently. With the development of scale pig industry, using of a single vaccine forimmunization is not only time-consuming and laborious, but aslo causes great stressto pigs. There is an urgent need to a combined inactivated vaccine to simplify theimmunization program for modern large-scale pig-breeding industry.. In this study, aPorcine circovirus disease-Japanese encephalitis-Porcine Parvovirus combinedinactivated vaccine was developed.1. An indirect ELISA was successfully developed based on PCV2Cap protein.Truncated PCV2orf2gene was amplified by PCR, then subcloned into theprokaryotic expression vector pET28a. The resulted recombinant plasmid wastransformed into BL21(DE3) bacteria. Interest protein was expressed by inducingwith IPTG at a final concentration of40μM,30℃, for3h. SDS-PAGE analysisshowed that the Cap protein had been expressed correctly with about24KDa andmainly as a soluble form. Western blotting confirmed that the protein has a goodimmune activity. An indirect ELISA was developed to detect anti-PCV2antibodiesusing the purified PCV2Cap protein as coating antigen. The optimum conditions ofELISA was as follow: antigen was coated at2μg/well,37℃1h then4℃overnight;serum sample working dilution was1:200, working dilution of HRP-labelled secondantibody was1:2000, incubation at37℃1h; TMB Substrate incubation time was15min; Serum sample was determined as positive when its OD450nm value isgreater than or equal to0.211and as negative when its OD450nm value is less than 0.211. The established method has good specificity, repeatability and accuracy. Onehundred and twenty pig serum samples were detected for PCV2specific antibody,the results showed that the method and the PCV2commercial kits have highcoincidence rate. This indirect ELISA method can be used for PCV2antibodydetection and epidemiological investigation.2. Development of Porcine circovirus disease-Japanese encephalitis-PorcineParvovirus infection combined inactivated vaccine. Parvovirus infection combinantinactivated vaccines and corresponding single vaccines were developed based on thePCV2XX strain, JEV XX strain and PPV XX strain. After the physical test, sterilityand safety test, KM mice immunized and BALB/c mice were immunized with thedeveloped vaccines. Antibody duration, effects of intramuscular and subcutaneousimmunization routes, and protection efficacy were evaluated. Immunogenicity andeffects of intramuscular and subcutaneous immunization routes onimmunologenicity were determined in guinea pigs. The results showed that after theimmunization with Japanese encephalitis related vaccines, anti-JEV antibody levelreached the peak at six weeks after the second immunization, better than thecorresponding single vaccine and bivalent vaccines, and fourteen weeks after thesecond immunization antibody level was still protective; four weeks after the secondimmunization, PCV2antibody level reached peak and was higher than that ofcorresponding single vaccine; two weeks after the second immunization, porcineparvovirus HI antibody titer in guinea pigs was protective, six weeks after the secondimmunization reached the peak, and it’s higher than corresponding single vaccineand bivalent vaccines; And the intramuscular immunization route is superior to thesubcutaneous immunization route. Challenge experiments showed that two weeksafter the second immunization, the triple inactivated vaccine can provide70%protection to KM mice against JEV Nj2008strain, and complete protection toBALB/c mice against PCV2XX strain. |
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