| Porcine Epidemic Diarrhea Virus (PEDV) and Transmissible Gastroenteritis Virus (TGEV) are major factor of swine viral diarrhea, and diarrhea of newborn piglets less than2weeks old caused by these two pathogens is particularly serious, which leads to severe vomiting, diarrhea and dehydration on infected piglets and nearly100%mortality rate, resulting serious economic losses for the swine industry. Thus, the enhancement of swine’s specific enteric mucosal immunity plays a vital role in the prevention and therapy for both PEDV and TGEV. Based on the clinically isolated and then identified PEDV, the shared pathogenic mechanism and immune characteristics of PEDV and TGEV are taken into consideration to study on the the pathogenic mechanism of the innovatively contrusted oral DNA vaccine that could enhance mucosal immunity efficiency:PEDV S gene and TGEV S gene are used as target genes and inserted into dual promoter of eukaryotic expression vector pVAXD to conduct and study the immunogenicity of PEDV and TGEV bivalent DNA vaccine delivered by attenuated Salmonella typhimurium SL7207. The main contents were as follows:1. Isolation and identification of Porcine Epidemic Diarrhea Virus (PEDV)In this study, PEDV Sichuan strain SC-L was successfully isolated from diarrhea fecal samples via Vero cells, which followed by preliminary study on its biological characteristics. The proliferation of PEDV SC-L on Vero cells was stable during serial passages and typical lesions appeares48hours post-inoculation, the cells get round, swell, and ultimately shrink, splid, and shed from the culture plate. Observed by transmission electron microscopy, the hollow or solid virus particles are scattered or in lattice-like arrangement in infected intracytoplasm; the virus particles pile or cluster on the cell membrane, fusing with the cell membrane and secreting outside the cell membrane. This RNA virus is insensitive to5-bromodeoxyuridine urinary nucleosides but sensitive to chloroform and aether, it loses infectbility when in the circumstance of60℃or above for 30min. By Sequencing S1domain of S gene, it is in the highest homology with South Korea DR13strains with nucleotide homology high up to99.2%. The phylogenetic tree analysis shows that the isolated strains and South Korea DR13isolated strains are in the closest genetic relationship. This successfully isolated strain accounts for material foundation of diagnosis and prevention for PEDV.2. Cloning and prokaryotic expression of PEDV S gene main antigtic domain fragmentThe PEDV S gene main antigtic domain fragment Sa were amplified from the PEDV S gene TA cloning plasmid via PCR and then cloned into the pMD19-T simple vector to construct the recombinant plasmid pMD19-T-Sa. The amplified gene is739bp in length and is able to encode247amino acids accroding to the sequence analysis. The recombinant prokaryotic plasmid pET-32a(+)-PEDV-Sa with Sa instered into pET-32a(+) prokaryotic expression plasmid could produce45Kd fusion protein in E.coli BL21, which is detected by SDS-PAGE electrophoresis. The expressed protein was purified by affinity chromatography immune and polyclonal antiserum was prepared by immunizing rabbits with this PEDV Sa recombinant protein.3. The construction of PEDV S gene and TGEV S gene co-expression eukaryotic plasmidThe RT-PCR amplified S1region of PEDV S gene that contains main neutralization epitope and receptor binding region of the virus, and5’fragment of S gene of the TGVE SC-H strain that covers A and B, and C, and D antigens points were respectively cloned into pMD-19T simple vector to construct recombinant of plasmid pMD19-T-PS1and pMD19-T-TS. The amplified PEDV S1gene and TGEV S gene were2367bp and1896bp in length, which respectively encoded789and632amino acids according to the sequence analysis of these two recombinant plamids. Sequence alignment analysis showed that the deduced amino acids from PEDV SC-L strain S1gene and TGEV SC-H strain S gene respectively shared88.8%-98.6%and95.5%-99.7%homology with other PEDV strains and TGEV strains. The eukaryotic plasmid pVAXD-PS1, pVAXD-TS and co-expression plasmid pVAXD-PSl-TS were constructed and tranfected into COS-7cells. Vitro expression of the recombinant plasmids were confirmed via indirect immunofluorscence assay, which indicated that the eukaryotic expression plasmids were correctly constructed and successfully displayed specific immunofluorscence after transfected into COS-7cells, demonstrating that the three contrusted plamids pVAXD-PS1, pVAXD-TS and pVAXD-PS1-TS could successfully express in vitro, these research offered a foundation on which to further develop PEDV and TGEV DNA vaccines.4. Oral immunization of attenuated Salmonella typhimurium harbouring PEDV S and TGEV S vaccineThe eukaryotic expression plasmids pVAXD-PS1, pVAXD-TS and pVAXD-PS1-TS were transformed by electroporation into attenuated Salmonella typhimurium SL7207to construct recombinant attenuated Salmonella typhimurium harbouring double-gene of PEDV and TGEV. After orally inoculated with a recombinant attenuated Salmonella on mice, we extract total cellular RNA from the terminal ileum of inoculated mice and detected the transcription of S1and S gene via RT-PCR. In safety analysis, the mice were orally inoculated with recombinant Salmonella at the dosage of1×109CFU per unit, the results showed that recombinant strain was relatively safe. Mice were orally inoculated at dosages of1×109CFU per unit of mixed recombinant strains SL7207(pVAXD-PS1) and SL7207(pVAXD-TS), and SL7207(pVAXD-PS1-TS) at the dosage of1×109CFU per unit, specific serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA. The results indicated that the recombinant attenuated Salmonella can provide good immunogenicity by effectively eliciting production of specified PEDV and TGEV serum IgG and intestinal IgA antibodies. Comparing the antibody levels between single-gene and double-gene immunity group, highest level of specific serum IgG and intestinal mucosal IgA antibody comes from single-gene immunity group. And was still significantly higher than (P<0.05) other two immunity group6weeks post-immunization. This study laid a foundation for the research and application of PEDV and TGEV oral DNA vaccine. |
PDF Full Text Request