Isolation And Identification Of Campylobacter Spp. And Prokaryotic Expression Of FlgH Protein Of Campylobacter Jejuni

Campylobacter is a gram-negative,arcuate,non-spore and fastidious bacteria.The bacteria can easily cause human bacterial gastroenteritis.Campylobacteriosis is the main foodborne disease in the world,Campylobacteriosis has the characteristics of short incubation period,high infection rate and long duration.Campylobacter is usually acquired by eating uncooked beef and drinking contaminated water and milk,leading to human foodborne diseases.however,overuse of these antibiotics has led to the emergence of antibiotic-resistant strains.So there will be resistance caused by Campylobacter infection,at the same time can lead to economic burden.Firstly,the source ofCampylobacter was isolated and identified in Ningxia by molecular microbiological methods.Then bioinformatics methods were used to identify candidate vaccine proteins.Samples of some rangelands in Ningxia were collected,including milk samples(136 samples),diarrhea cow dung samples(32 samples),and intestinal tubes(6 pieces);Through isolation and culture,biochemical test,PCR detection and sequencing,identification of bacterial and virulence gene detection.Three strains of Campylobacter jejuni(C.jejuni)were isolated from 136 milk samples,and the positive rate was 2.2%.Four strains of Campylobacter coli(C.coli)were isolated from feces samples of diarrhea with a positive rate of 12.5%.This study used Vaxign,VaxiJen v2.0,and BCPreds software to predict candidate vaccine antigens.The main criteria for selecting immune proteins are protein localization,antigenicity,and B-epitopes.Twenty proteins were tested for potential vaccines.Four new proteins(two extracellular proteins,FlgK and FlgE;two extramembrane proteins,FlgH and hypothetical protein,Cj1252)were found to be suitable for vaccine research.These newly identified antigens then need to be tested in vitro and in vivo to demonstrate their immunity and protection.From the candidate vaccine proteins were identified above,The prokaryotic expression and immunogenicity analysis for preselect flagellin FlgH.Amplified full-length primers for the FlgH gene were designed and PCR amplification using C.jejuni standard strain 11168(NCTC 11168)as a template,the purpose of the fragment with the vector plasmid pET-32a(+)were connected;Recombinant plasmid pET32a(+)-FlgH was transformed into competent cell BL21,then induced by IPTG and identified ppropriate induction concentration and induction time,SDS-PAGE analysis and finally purified protein.His-FlgH purified protein was immunized 6-week-old ICR mice by three routes of vaccination(dorsal asubcutaneous,leg muscle,and peritoneal cavity)and serum was collected after immunization.The indirect ELISA was used to detect the multi-antiserum titer,Western bolt analysis showed that the multi-antisera could specifically bind to the recombinant protein His-FlgH.As a result,the titer of the multi-antibody serum in the subcutaneous and muscle groups was 25,600 which was greater than the titer of 12,800 in the multi-resistance group of the abdominal cavity group.The results can prove that FlgH has a certain biological activity,so further proof of its potential as a subunit vaccine,it also provides a theoretical basis and experimental basis for the next step in the application of the protein vaccine to host and test its immunoprotection.The Campylobacteriosis vaccine is an intervention that prevents Campylobacter from infecting humans and host animals,thereby effectively reducing the incidence of disease in animals.