Research On Immunogenicity Of Attenuated Salmonella Harboring S And N Genes Of Transmissible Gastroenteritis Virus(TGEV) In Mice

Transmissible gastroenteritis(TGE) causes serious intestinal infectious diseases in piglets, resulting in great economic loss all over the world, and is especially severe in newborn pigs less than two weeks old, causing nearly100%mortality. Until now, the measures to treat it is not ideal.Transmissible gastroenteritis virus, which belonging to cornavirus, has a positive single-stranded RNA about28.6kb and consists of three major structural proteins:a phosphorylated nucleoprotein (N) and two glycoprotein, the membrane(M) and spike (S) protein.The S and N protein,especially the S protein, is a candidate for novel vaccine. In this study, we used attenuated S. typhimurium SL7207as carrier for delivery of DNA vaccine, which encoding the N and N-terminal of TGEV S gene in dual promoter plasmid pVAXD, via intragastric way to study the immune effect.1. Transformation of pVAXD-N-Sa to attenuated S. typhimurium strain SL7207and Detection of stability, safety of the recombinant vaccine in vitro and vivo.The plasmid pVAXD-N-Sa was electroporated to attenuated S. typhimurium strain SL7207,the positive transformants verified was named SL7207(pVAXD-N-Sa).Then, determine the stability of SL7207(pVAXD-N-Sa) in vitro, detect the transcription of S and N gene in vivo, check the bacterial colonization in liver and spleen and detect its safety in vivo. SL7207(pVAXD-N-Sa) was cultured with and without Kanamycin selection, Plasmid stability was determined by count the viable bacterial number. Three days after immunization, Payer’s patches were removed from mice, the transcriptional of TGEV S gene in Payer’s patches was analyzed by RT-PCR using specific primers. Three groups of six-week-old BALB/c mice, with18mice in each group, were inoculated with SL7207(pVAXD-N-Sa) at dosages of5×108,1×109, and2×109CFU per mice, and boosted with the same dosage two weeks later. Spleens and livers were collected at week1,2,4,6,8.The bacterial counts were determined by plating homogenized spleen and liver samples on agar.In our present study,the results showed that the plasmid in selective medium was present in50%after100generations,no bacterial retained the plasmid after approximate100generations.the RT-PCR products of500bp in size for S and400bp for N confirmed that the S and N gene could express at3days after the inoculation with SL7207(pVAXD-N-Sa).Groups of mice were immunized orally with SL7207(pVAXD-N-Sa) at dosages of5×108.1×109,2×109CFU per mice. Between weeks1and2, the bacterial numbers decreased in both the liver and spleen. Recovery of SL7207(pVAXD-N-Sa) from livers and spleens of mice orally immunized with5×108,1×109,2×109CFU,the numbers rose slightly during weeks2and4(the first booster immunization was at week2), but fell rapidly in the livers and spleen, and no recombinant bacteria were isolated from the liver or spleen weeks8post-immunization.2. Preparation of the antigens which used for antibody detectE.coli cells of the strain BL21(DE3) were transformed with plasmids pET-32a-SBC and pET-32a-N.For Bc and N expression and purification, they were induced with IPTG. Then, recombinant proteins were purified using Ni-NTA according to manufacturer’s specifications.The results showed that bands of38KD and65KD were observed in the cases, indicating that SBC and N protein expressed in the right way, as expected.All the purification steps were followed by western blot analysis with specific antibodies.Observations showed that the reactionogenicity of SBCand N protein was nice.3. Detection of humoral, mucosal and cellular immunity of recombinant vaccinesThe mice were orally fed with SL7207(pVAXD-N-Sa),SL7207(pVAX-S),SL7207(pVAX-N) and SL7207(pVAX-S-N) as immunized group, SL7207(pVAXD) and PBS as control group, at the dosage of109CFU per mouse. All groups were boosted immunized two times with an equivalent dose with2weeks intervals after the initial immunization. Total serum IgG and IgA levels of immunized mice were measured by ELISA, while using the purified TGEV S and N protein as coating antigen. Proliferation of peripheral blood mononuclear cells and Spleen lymphocytes were detected by MTT. The serum IFN-γ or IL-4levels were analyzed with an IFN-γor IL-4detection kit according to the manufacturer’s instructions.The results showed that specific IgG and IgA were observed in all vaccinated mice at week2-8. SL7207(pVAXD-N-Sa) could elicited specific anti-S protein and specific anti-N protein local mucosal and systemic immune responses. At week2post-immunization specific anti-S protein IgG was detected in mice immunized with SL7207(pVAXD-N-Sa),at week6post-immunization, the IgG levels reached peak, at week8post-immunization, the IgG levels decreased.Immunizations with immunized group could elicit recognizable levels of antigen-specific T cell responses, and compared to splenocyte, the level of lymphocytes in peripheral blood mononuclear cells was stronger. The proliferation of T lymphocytes in the blood and spleen of mice immunized with SL7207(pVAXD-N-S), was increased during weeks2-8post-immunization, and the peak was generated at week6.Levels of IFN-y and IL-4were produced when immunized with immunized group, when investigate the IFN-y produced by serum of vaccinated animals,the IFN-y peak level was at week6, then the level of IFN-y was decreased at week8.when investigate the IL-4produced by serum of vaccinated animals, no significant differences were observed comparing the group receiving SL7207(pVAXD-N-Sa),SL7207(pVAX-S-N),SL7207(pVAX-S),SL7207(pVAX-N) and the control groups,which receiving PBS and SL7207(pVAXD)(P>0.05).Our results showed the SL7207(pVAXD-N-S) preserve good oral immunogenicity, and could induce comprehensive immune responses.