Research On Recombinning Of Salmonella Flagellar Genes With Three Serotypes And Immunologic Detecion

Food poisoning badly threats the health of human of which the chief reason is bacteria pollution,while the leading pathogen to cause food contamination is salmonella,so a rapid and simple detective system is necessary for the etilolgic diagnosis of salmonella.This experiments chosed three virulent strains of salmonella: salmonella paratyphi A, salmonella typhimurium and salmonella choleraesuis, serotype ofⅠflagellin of which is a, i and c respectively.Referred to the genomic sequences ofⅠflagella deposited in the GenBank datebase,three pairs specific primers were designed.The primers were specially for the determinant encoding genes of the three serological flagella antigens which is H1-a,H1-i and H1-c respectively.Fragments of interest genes were amplified by polymerase chain reaction.The fragments of Fa,Fi,Fc was 1088 bp,633bp,1056bp respectively.Products of PCR were ligated to the pMD-18T vectors and recombination plasmids were constructed successfully.The right recombination plasmids named pMD-18T-Fa, pMD-18T-Fi,and pMD-18T-Fc.In this foundation,the recombination plasmids was tranfromed into E.coli DH-5αand plasmids were extracted and digested by restriction enzymes.The sequenced results indicated that the nucleotide sequences of interest genes were consistent with the objective sequences completely.According to the principle of Genetic Engineering,the primers including two linkers were designed to splice the three genes of flagellar antigen to recombination gene,the length of which was 2747bp in all.Products of PCR were ligated to the pMD-18T, sequences of interest gene in the recombination plasmid was consistent with the sequences of objective gene in 99 percents.The recombination plasmid pMD-18T-F was constructed successfully. Constructed prokaryotic expression vectors pET-28a-F of flagellar recombination gene,transformed it into Escherichia coli BL21.Sieved and identified Escherichia coli engineering strains of pET-28a-F-DE3,which expressed interest protein.The purpose protein were expressed by IPTG-induced engineering E.coli strains,SDS-PAGE electrophoresis results showed that the size of the fusion flagellin is 95Ku.pET-28a-F-DE3 induced in different conditions expressed interest proteins differently. SDS-PAGE of proteins indicated that maximum of interest protein were got at the condition:the final concentration of IPTG was 1mmol/L, cultivated for 6h at 37℃.The thin-layer scanning analysised that interest protein accounts for the 11.2 percents of all expressed proteins, when most of the interest protein was existence in the form of inclusion bodies.Bacterium cells were broken by using ultrasonic wave,the cytorrhyctes included interest protein were extracted,purified,and dissolved. Concentration of solution of the inclusion protein measured by UV spectrophotometer was A280=3.25mg/mL.The cytorrhyctes was immunogen for the immune animal experiments.The fusion flagellin were purified by reclaimming gelatum,electric elution and dialysis.The concentration of depurated fusion protein was A280=0.209μg/μL.The depurated fusion protein was used to detect antibodies in the indirect ELISA test. Per Guinea pig was immuned 500～800μg inclusion proteins to acquire serum antibody. Antibody titer measured by agar diffusion test was: titer of antibody reacted to fusion flagellin was highest at dilution by 16 multiples.Antibody titer tested by indirect ELISA was achieved 1:512000, and the sensitivity of antibody responsed to fusion flagellin was 8μg/mL.Salmonella paratyphi A, salmonella typhimurium and salmonella choleraesuis could be detected by serum antibody which diluted by 10,000 multiples, at least:1.35×105/mL, 1.63×105/mL,2.31×105/mL. The results of indirect ELISA test demonstrationed that serum antibody reacted with objective salmonella strains specially and cross-immune reaction didn′t happen when serum antibody were togerther with other fifteen kinds of bacteria strains caused food poisoning.Serum antibody were purified by OctanoicAcid-ammonium sulfate precipitation,the concentration of purified protein measured by spectrophotometer was A595=3.147mg/mL. The preliminary detection method of colloidal gold indicator paper was established.This experiments did groundworks for the further application of researches,as well as for the establishment of broad-spectrum, rapid detection methods in food poisoning. In this experimental study,flagellar genes which encoded three serotypes antigens were reorganised. Expressed fusion protein maintained three original antigen characteristics of salmonella flagellar,and polyclonal serum antibody against the epitopes were obtained. So the researches was implement for the rapid detection of many flagella serotypes of salmonella,offering new thinkings for developing inspection techniques of food hygiene.