[Construction of recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis spike glycoprotein and analysis of immunogenicity].

Transmissible gastroenteritis virus (TGEV), is an enteropathogenic coronavirus that causes a highly fatal acute diarrhea in newborn pigs. It's typically clinical manifestations consist of omitting, severe diarrhea, loss water and highly infectious disease. All kinds and ages of pigs can be infected. Particular, the mortality piglets under 3 weeks may reach 100% . The effective protection against TGEV requires the development of vaccines that are able to induce local mucosal immunization. Lactococcus lactis was selected as a bacterial carrier for the expression of TGEV spike glycoprotein. The gene of S glycoprotein was cloned into the Lactococcus lactis vectors pNZ8112. An approximately 2000 bps fragments of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in the recombinant strain pNZ8112-Sa/NZ9000. The recombinant glycoprotein S was detected by SDS-PAGE and Western blot after induced by 1ng/mL nisin. The result indicated that the expressed product maintain the antigenicity of TGEV as expected. In order to detect the location of expressed protein, the yellow and green fluorescence of the recombinant strain pNZ8112-Sa/NZ9000 was detected by the IFA experiments, which indicated that the expressed recombinant protein was secreted and located on the surface of the bacterium cell. Oral immunization of BALB/c mice with recombinant strain that constitutively express the 66kDa fragment of the glycoprotein S, Specific anti-TGEV glycoprotein S secret immunoglobulin A (sIgA) antibodies were detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces after immunization. It was showed that the mice immunized with pNZ8112-Sa/NZ9000 recombinant strain had produced clear antibody level anti TGEV, and which had provided important substance foundation and explored the feasibility of Lactobacillus as oral vaccine.