Development Of Monoclonal Antibody Against Salmonella PEG Fimrbrae And Preliminary Clinical Application

Salmonella is widely distributed in nature, which is Gram-negative bacteria, and an important zoonotic pathogenic bacteria in Enterobacteriaceae genus. The confirmed Salmonella serotypes have been up to more than 2600 serotypes currently. In livestock’s production and breeding, Salmonella contamination has been a nonnegligible problem, in which S.enteritidis and S.gallinarum-pullorum are the dominant serotypes of Salmonella contamination in poultry breeding, causing the decrease of production performance of laying hens, gradual weight loss of broilers, and large number of chicken deaths, etc., which cause huge economic losses to the breeding industry.This article researches on PEG fimbriae specially expressed in S.enteritidis and S.gallinarum-pullorum in Group D Salmonella, amplifies the fimbriae’s main subunit pegA gene through PCR, builds prokaryotic expression system and prepares monoclonal antibody of PEG fimbriae, takes advantage of slide agglutination to rapidly detect of S.enteritidis and S.gallinarum-pullorum, and provides basis for judging the dominant serotypes of poultry Salmonella contamination. This article can be divided into three parts:Part one:The article discusses the prokaryotic expression of the main subunit gene pegA of Salmonella PEG fimbriae. According the complete genome sequence of PEG fimbriae in GenBank, the article designs a pair of specific primers for the main subunit pegA, takes S.pullorum standard strain 526 genome as a template, and takes advantage of PCR amplification technique to largely obtain target genes of pegA. The gene fragment is cloned into pMD19-T vector, and the sequencing result shows that the sequential size is 531 bp, conforming to the expected size and no mutation in sequence. To prepare and purify recombinant protein, it clones pegA gene fragment into the prokaryotic expression vector pET-22b(+) labeled with HIS, and constructs the recombinant plasmid pET-22b (+)-pegA. It converses the screened recombinant plasmid pET-22b (+)-pegA into E.coli BL21 (DE3) competent cells, carries out induced expression, and obtains the soluble protein with a size of about 23 KDa. After the purification of Ni-TED 2000 kit, the expressed recombinant protein obtains purified recombinant protein with the concentration of about 0.83 mg/mL, which is designated as rPegA. It analyses the size and reaction specificity of protein band through SDS-PAGE and Western blot, and determines the prepared fusion protein as the prospective objective protein.Part Two:Preparation of anti-salmonella PEG fimbria monoclonal antibody, apply lymphocyte hybridoma technique to immune BALB/c mice with the expressived recombinant protein and obtain three plants of stably excretive monoclonal hybridoma cell strain named as ID 10,3D 12 and 4B4 respectively. These three hybridoma cell strains revive through freeze of the liquid nitrogen with no declining of titer after continuous passage showing good stability. Use indirect ELISA method to test McAb ascites titer of 3D 12 to be 1:64000. The test result of Western blot shows that the preparative monoclonal antibodies can all specific react with recombinant protein, S.enteritidis CMCC 50336, S.pullorum CVCC 526 and S.gallinarum T63, but all cannot react with S.typhimurium T14 or E.coli DH5a. The result of slide agglutination test shows that McAb ascites have agglutination with S.enteritidis CMCC 50336, S.pullorum CVCC 526 and S.gallinarum T63 to positive reaction but have no agglutination with S.typhimurium T14 or E.coli DH5a. It indicates that PEG fimbria monoclonal antibody prepared in this experiment has good specificity.Part three:The article carries out the preliminary clinical application of monoclonal antibodies of Salmonella PEG fimbriae. Builds slide agglutination test on monoclonal antibodies of PEC fimbriae. If the result is positive, it indicates that the checked bacterial liquid contains S.enteritidis and S.gallinarum-pullorum, on the contrary there is no. Taking monoclonal antibodies of PEG fimbriae as detection antibody, it carries out slide agglutination test to detect 72 poultry Salmonella separately preserved in the laboratory and detects 43 positive samples; regards the diagnostic serum of Salmonella as a parallel control, and detects out 45 positive samples with S.gallinarum-pullorum and S.enteritidis. The coincidence rate of Salmonella diagnostic serum and slide agglutination test result of PEG monoclonal antibodies is 95.6% (43/45). The slide agglutination test method mediated on monoclonal antibodies of Salmonella PEG fimbriae developed in this experiment has potential application values in detection and purification of S.enteritidis and S.gallinarum-pullorum in poultry breeding industry.