| Porcine epidemic diarrhea (PED) is an acute disease of swine caused by porcine epidemic diarrhea virus (PEDV). It's featured with acute enteritis and watery diarrhea. Mortality in infected neonatal piglets can approach 95% because of acute enteritis and severe watery diarrhea. Porcine epidemic diarrhea has been widely distributed in the world, causing serious economical loss. The secretory IgA antibody generated by stimulating enter-mucosal immunoctes is an effective antibody in preventing the PED. It is important to develop a new vaccine which can induce efficient systemic and mucosal immune responses after vaccination.It has been identified that the partial protective gene of S gene (COE gene, 499-638aa) is the main gene which can induce the body to produce neutralizing antibodies. Additional, the SD domain of S1 subunit contains a linear epitope (697-742aa) and two B cell epitopes (744-759aa and 756-771aa) which are both excellent candidate antigens for making vaccines.As a vaccine vector, the attenuated Salmonella typhimurium can submit antigen effectively, which can induce of systemic immunity and mucosal immunity stimulated by the Salmonella typhimurium and exogenous antigen.In view of the reasons above, we expected to constructed a recombinant attenuated Salmonella typhimurium, which took attenuate strain of Salmonella enterica serovar Choleraesuis absenting asd C500 as vector, PEDV COE gene and SD gene as the target genes. We also want to determined the immunogenicity of the recombinant strain. And expected to develop a vaccine which can prevent the porcine epidemic diarrhea effectively.1 Construction and identification of recombinant attenuated Salmonella enterica serovar Choleraesuis strainsAccording to the reported sequence of Porcine epidemic diarrhea virus strain CV777, we selected two fragments of the spike gene—OE gene and SD gene as the target genes. About 480 bp gene fragments (COE) and 462 bp gene fragment (SD) were amplified by PCR from the feces of piglets suffered from porcine epidemic diarrhea disease. Sequence alignment analysis indicated that the homogeneities between COE gene and SD gene from PEDV strain CV777 were both reached 97%, and had more than 91%homology in different PEDV strains published. Then we cloned the target gene into the pYA3493 vector, and constructed non-resistance expressing plasmids pYA-COE and pYA-SD. At last, we constructed recombinant Salmonella strain C501-COE and C501-SD by electrotransformating the plasmids into C500 competent cells. The results of identification showed that we have constructed the recombinant Salmonella strains successfully.2 Biological characteristics of recombinant strains C501-COE and C501-SDWe identified biological characteristics of recombinant Salmonella strains, including the expression level, the growth and phenotypic characteristics, and the hereditary stability. The expression and localization of the recombinant protein were detected by Western blotting and indirect immunofluorescence. The results of Western blotting and indirect immunofluorescence showed that the 17.8 kD and 17 kD fusion proteins were expressed in lysates of C501-COE and C501-SD separately, and the expressed proteins of the recombinant strains located on the surface of the Salmonella. Besides, the recombinant Salmonella strain didn't change their grouth characteristics. After incubating 11 hours at 37℃, the OD600 of parental strain and recombinant strains could reach to 1.0. The identification of phenotypic characteristics and hereditary stability showed that the recombinant Salmonella strains had the same biochemical characteristics as parental strain C500, and the exterior protein could be amplifyed from the recombinant strains after propagating to 50th generation, and sequence analysis also showed that the sequences of the exterior genes remained unchanged.3 The immunogenicity efficacy of the recombinant Salmonella strains in mice.The immunogenicity efficacy of the recombinat stain C501-COE and C501-SD as new engineering vaccines were evaluated using a mouse model. Mice were orally immunized and intramuscular injection with C501-COE and C501-SD at the dose of 1.5×108 CFU, and then we tested each immunological index with the method of ELISA and fluorescence quantitative PCR. The results showed that the orally immunized groups of recombinant strains C501-COE and C501-SD could induce high IgA titers with 1:1280 and 1:2048, which higher than the intramuscular injection groups (1:640 and 1:1024), but, the intramuscular injection groups could induce IgG titers with 1:4096 and 1:2560 higher than orally immunized groups. And intramuscular injection groups could induce more IFN-y after stimulating the splenocyte of mice in vitro.than the orally immunized groups.So these two different inoculation ways could not only induce mucosal immunity and humoral immunity, but also cellular immune response as well, and better mucosal immunity was induced by administered orally.4 The initial clinical applications of the recombinant Salmonella strain in porcine.In order to observe the effect of the recombinat vaccines, we carried out clinical trials. At one day of age, piglets orally immunized with the minture of two recombinat strains at the dose of 3 X 109 CFU, the results showed that the recombinat vaccine safety was better and no severe adverse effect was encountered. The rates of mortality and diarrhea reduced a lot after orally immunized suspected piglets of porcine epidemic diarrhea with recombinat vaccine. The statistical result showed that the mortality rates had fallen from 80% to 20%～50% and the rate of diarrhea had fallen from 100% to 18%～30%. The ressults of clinical trials showed that the recombinat vaccine had a well prophylactic effect. |
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