Studies On Immune Effect And Preparation Of Recombinant Lactobacillus Plantarum Expressing OmpC-DC Against Salmonella Pullorum

Pullorum Disease is an acute septic infectious diseases with white feces caused by pullorum Salmonella. Disease spreads in all ages of chicken,which widely distributed in the domestic chicken farm, the infection rate is higher in the poultry industry, one of the important infectious diseases. This experiment proved Salmonella outer membrane protein having a good immune protective effect, OmpC is a porin protein of the outer membrane proteins, which can be mediated by humoral immune response and also has an important role in cellular immunity. In recent years, OmpC used in vaccines and high titers of antibody as an adjuvant or immune stimulating agent more and more. Dendritic cell-targeting peptides(DCpep) are peptides with36bases, specific targets and combines to DCs, guides and regulates the specific immune. PCR amplification of the OmpC gene from pullorum Salmonella virulent strain CVCC528, connected to the carrier pSIP-409of E. coli-lactic acid bacteria shuttle expression, it was expressed in both E. coli and recombinant Lactobacillus.Take the genetic engineering of lactic acid bacteria feeding chicken, it made good immune effect. And specific research contents are as follows:In this research, PCR primers were designed according to OmpC gene sequences published in GenBank, OmpC gene was amplified pullorum CVCC528genome as a template, and the introduction of DCpep and OmpC gene fusion, the PCR products were cloned into pGEM-T vector, and transformed into E. coli competent cells DH5α. Through plasmid extraction, PCR identification, double digestion, sequencing and analysis methods,results showed that it was success to cloned the OmpC gene with DCpep. And BLAST analysis showed100%homology.OmpC and OmpC-DC gene, which cloned into pSIP-409and transformed into E. coli competent cells BL21, after identified successfully, it transformed into Lb.plantarum NC8to constructed recombinant plasmid, SDS-PAGE and Western blotting analysis showed that, OmpC-DC expression of the protein has reactogenicity successful.Designed immunization program, the recombinant genetic engineering of lactic acid bacteria fed1-day-old chicken, through the methods such as changes in body weight, lymphocyte proliferation assays, ELISA with detect serum antibody IgG and intestinal contents SIgA, the results showed that the recombinant lactic acid bacteria pSIP-409-OmpC and pSIP-409-OmpC-DC produced a good immune effect. And the recombinant lactic acid bacteria with DCpep effected better than the separate recombinant lactic acid bacteria pSIP-409-OmpC.This experiment made pullorum Salmonella OmpC gene with DCpep expressed in lactic acid bacteria successfully, immune to good effect, to lay the foundation for the development of the recombinant pullorum gene engineering of lactic acid bacteria vaccine.