The Surface-Displayed TGEV-S Epitopes On Lactobacillus Casei And Analysis Of It's Immunogenicity

Procine transmissible gastroenteritis virus(TGEV) causes serious diarrhea in piglets with high mortality.It is an important disease for the development of pigs.In order to produce a viable organism expressing antigens for safe and cheap.The BC and A epitopes of TGEV were amplified using the TGEV-S cDNA as template and subcloned into the multiple cloning site of expression plasmid pQE-30xa between BamHI and SalI.The recombinant plasmid which had correct sequence and ORF by identification of endonuclease digestion and sequence analysis was named pQE-BC and pQE-A ,then transformed into E.coli competent cell XL1-blue and induced with IPTG,the fusion proteins were expressed at high level.The expressed protein could be recognized by antibodies of Anti-His-Tag and anti-TGEV using western blot method. The recombinant protein was highly purified by Ni-NTA agarose and save at -20℃as diagnostic antigen. The BC and A genes were disgested from pQE-BC and pQE-A vector, respectively. They were fused to the surface-displayed vector pLA which has a surface antigen display system for lactic acid bacteria using the poly-r-glutamic acid synthetase A protein (pgsA) as an anchoring matrix. The recombinant vectors pLA-BC and pLA-A were transformed into Lactobacillus casei by electroporation. Approximately 60kD and 58 kD fusion protein were detected on surface of Lactobacillus casei by SDS-PAGE, Western-blot, indirect immunofluorescence microscopy and flow cytometry. The feces and serum samples were collected from BALB/c SPF mice which were orally and nasally immunized with recombinant Lactobacillus casei on different days.Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides.To better characterize antibody responses against s protein fragments, the levels of antigen-specific IgG subclasses(IgG1, IgG2a ,IgG2b, IgG3)and other antibody isotypes IgA and IgM were assessed by ELISA.Both intranasally and orally immunized mice developed s protein specific antibodies that were predominantly IgG2b with moderate levels of IgG2a and IgG1. The subclass ratio calculated as IgG1/(IgG2a+IgG2b) was less than 0.4 indicating a Th1 type response.