Construction Of Live Genetic Engineering Vaccine Against Transmissible Gastroenteritis And Their Immunogenicity Efficacy

Transmissible gastroenteritis virus (TGEV) is the etiological agent of transmissible gastroenteritis(TGE), which is a highly contagious enteric disease in piglets causing high mortality rate of up to 100% and resulting in severe economical losses to the inffected farms. Current TGEV vaccines can not provide effective immune response to piglets which emphasizes the importance of developing new vaccine which can prevent and control this pathogen effectively. How to enhance expression of main antigen sites is a crucial thing. Site A as well as site D in the spike protein (S) are the main inducer of neutralizing antibodies and the previous is a major B cell epitope. Peptide N321, which are located on the nucleoprotein (N), defines a functional T helper epitope eliciting T cells capable of collaborating with B cells specific for different proteins of TGEV. In this work, we cloned and integrated the synthesized genes (SLN) into non-resistance expressing plasmid pYA3493, and successfully constructed recombinant Salmonella strains in which can express the fragments integrated efficiently. Besides, by using a vaccination and challenge trial both in mice and in piglets, we examined the protective efficacy and immunogenicity of the recombinant Salmonella strains.1. Construction of recombinant Salmonella enterica Serovar Choleresuis strains.Through the analysis of bioinformatics, we notice that antigen site A as well as D in the spike protein (S) are the main inducer of neutralizing antibodies and the previous is a major B cell epitope; antigen site N321 in the nucleoprotein (N) defines a functional T helper epitope. Therefore, antigen site A, D and N321 of TGEV combined by two Linker ((GGGGS)3) as a fusion antigen epitope (SLN) synthesized in company. By appling the socaudarners (SalI and XhoI), two copy of SLN (2SLN) was constructed on plasmid pET-28a. Then non-resistance expressing plasmids pYA-SLN, and pYA-2SLN were constructed. Recombinant Salmonella strains C501-SLN and C501-2SLN were constucted by electrotransformating the plasmids into C500 competent cells. The recombinant Salmonella strains did not change their biocharacterics, even though highly expressing heterologous fragments. Recombinant strains C501-SLN and C501-2SLN could got back the ability to grow in DAP" LB culture and highly expressed the gene integrated in. The study of phenotypic and biochemical characteristics showed that these two recombinant strains were the same as parental strain C500. Genetic characteristics of study showed that the recombinant strains had the ability of a stable genetic.2. The immunogenicity efficacy of the recombinant Salmonella in mice. The immunogenicity efficacy of the recombinat stain C501-2SLN and C501-SLN as new recombinat vaccines were evaluated using mouse model. The results showed that the recombinant Salmonella strains C501-2SLN and C501-SLN could induce high titers of IgG and IgA against the antigens of rSLN and Salmonella. However, the recombinant Salmonella strain C501-2SLN could induce higher titers of IgG and IgA than C501-SLN in immunized mice. Furthermore, the peptide antigen containing two copies of the peptide produced significantly higher virus-neutralization titers, higher than single copy of SLN, indicating that a peptide antigen displaying repeating copies of SLN is a more potent inducer of TGEV-neutralizing antibodies than SLN alone, and may be useful for the production of specific neutralizing antibodies for active immunotherapy of TGEV infection. Besides, IgG subclasses were induced in mice immunized by C501-2SLN with the IgG1 titer, similar to IgG2a at second weeks after the second immunization, indicating that the recombinant Salmonella strains C501-2SLN could not only induce humoral immunity and mucosal immunity responses, but cell mediated immunity response.3. The immune and protective assay of TGE oral vaccine in pigletsAt 1 day of age, piglets orally immunized with 5.3×109 CFU C501-2SLN elicited rSLN specific serum IgG antibodies, albeit at low titer, and gut mucus secreted IgA within 7 days. Induction of active immunity was assessed by. challenge of pigs with virulent TGEV 1mL (1×106 TCID50/mL) at 7 day of age. Salmonella-expressed TGEV proteins induced mesenteric lymph node immune responses associated with IgA antibodies to TGEV and fully protection against TGEV challenge. The higher titers of serum IgG and gut mucus secreted IgA antibodies to TGEV in this group can be observed after TGEV challenge. Simultaneously, piglets survived the challenge with TGEV compared with 6 of 6 piglets in the vector-treated controls group and 6 of 6 piglets in the phosphate-buffered saline (PBS) treated controls group showed clearly clinical TGE symptoms. It indicatated that the recombinant C500-2SLN vaccine showed the potential as a new oral vaccine against TGEV infection.