Biological Characteristics And Immune Effect Analysis Of Salmonella Enteritis NmpC Gene Deletion Strain

Salmonella enteritis is a kind of intestinal intracellular bacteria,which can cause gastroenteritis and diarrhea,and can cause death in severe infections.Salmonella is a serious zoonotic bacteria.Antibiotics are often the first choice to control Salmonella.In recently,Salmonella is resistant to antibiotics and cannot be effectively treated.Vaccination is a good control measure.Homologous recombination technology was used to knock out the target gene,and the gene was preliminarily studied to provide reference for the development of live attenuated vaccine.In this study,the recombinant plasmid pET-28a-nmpC was obtained by molecular biotechnology.The recombinant plasmid pET-28a-nmpC was transformed into the expression strain BL21 and induced by IPTG.His-NmpC prokaryotic protein was obtained.SDS-PAGE analysis and Western blot validation showed that the purified NmpC prokaryotic protein was used to detect its immune protection and antibody level in immunized mice,and was identified by Western blot and ELISA.SDS-PAGE analysis showed that NmpC was expressed as inclusion body and its molecular weight was about 38 kD.Western blotting analysis showed that NmpC prokaryotic protein could be specifically identified with His labeled antibody and multi-antibody serum,indicating that NmpC prokaryotic protein had good immunogenicity.The immune protection of NmpC protein in mice was up to 70%,It is a good vaccine target.The chloramphenicol-sensitive pKD3 plasmid was used to replace the nmpC gene in Salmonella enteritidis genome by?-Red homologous recombination technique.The deleted nmpC gene of Salmonella enteritidis was detected by PCR;The C50336?nmpC was reconstructed by pBR322,and the immune NmpC protein was used.The serum was proved to be an anti-Western blot,and the nmpC gene deletion strain?C50336?nmpC?and complement strain of Salmonella enteritidis?C50336?were successfully constructed.The biochemical charateristics were tested between Salmonella enteritidis wild type strain and nmpC gene knockout mutant and teplenished strain.The results showed that the mutant was no difference in growth and biochemical characteristics.It also affects the synthesis of curl and cellulose protein;we tested the medicines,and bacterial virulence of Salmonella enteritidis wild type strain and nmpC knockout mutant and replenishment strain.The results showed that with medicines was reduced in nmpC gene knockout mutant,and virulence factors were also reduced in nmpC gene knockout mutant,the virulence of the deleted strains increased nearly 20 times than that of the wild type LD₅₀,and the virulence decreased.To evaluate the potential of deleted strains as vaccines,it was found that the protective rate increased by 20%in mice after immune safety dose deletion.This indicated that C50336?nmpC had the potential to be used as attenuated vaccine of Salmonella enteritidis and provided reference for clinical application..