A Preliminary Study On Using Of In Vivo-Induced Antigen Technology For Identification Of Escherichia Coli O139 Proteins Expressed During Piglet Infection

Porcine colibacillosis is one of the more serious infectious diseases in large-scale pig-raising industry, bring about huge economic losses to the pig farming industry.On the one hand,serotype of E.coli is complex,pathogenic Escherichia coli serotype is changing,resulting in immune failure,to the control of pig colibacillosis difficulties increased.On the other hand,in recent years a wide range of antibiotics and inappropriate use has led to resistant strains of E.coli growing,ever-changing drug-resistant mechanisms, the treatment of E.coli disease has become very difficult.Therefore,in this study,IVIAT was employed to find a few ivi genes of E.coli and to analyze their functions,and further more,to understand its molecular pathogenic mechanism,and to find a few clues for the development of new vaccine,diagnosis reagent and drug.In vivo induced antigen technology(IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in animal infections.The general principle is that the sera from naturally infected animals and absorbed with bacteria grown in vitro were hybridized with blotted colonies containing the gcne libraries of the pathogen.The positive colonies contain the genes which could be specially expressed in vivo and might be potential targets to novel vaccines,drugs and diagnostic methods.Notably,pathogenic immunogens expressed in vivo specifically induced by the pathogen infection have been thought to be important for the pathogenicity of the pathogen.To characterize potential virulent factors expressed by E.coli during the E.coli infection,in this study,use of in vivo induced antigen technology,conducted a study of the pathogenic Escherichia coli O139 of piglets,to screen antigens induced from Escherichia coli O139 of piglets in vivo.The first a genomic library of Escherichia coli O139 was constructed:pET-32a(+) expression vector was cleaved by BamHI,the linear vectors were obtained and de-phosphatized by CIAP.The E.coli O139 was cleaved by Sau3AI,1～4Kb DNA fragment was recovered.Then they were ligated into the prepared pET-32a(+) expression vectors,the recombinants were transformed into E.coli DH5a,Alkaline Lysis recombinant plasmid Prep,Recombinant plasmid was cleaved with Two Restriction Enzymes(XhoI and NcoI) for identification,the identification was in accord with requirement.A bulk of abstraction of recombinant plasmids were transformed into fresh competent cell BL21,to compute CFU of expression library,its capacity was 1.2×10~1 clones,which was enough for this study.Random picking 10 clones were cleaved with two above-mentioned Restriction Enzymes for appraising the library,these 10 clones are all possible clones.Restriction Enzyme cutting vector fragments were about 5.9kb,inserts were between 2Kb and 4kb.The construction of the genomic expression library of E.coli O139 was successful, and paved a firm way for the screen of the library.And then the genomic library of E.coli O139 were screened:Convalescence stage sera were pooled from pathogenic E.coli-infected piglets,the sera were check with SAT,compared with the coincidence rate,the coincidence was 30%,was consistent with tostrain serotype.These sera were absorbed with the lysate of E.coli O139 grown in vitro and the lysate of E.coli BL21.Use of ELISA for determination of the best dilution of sera and detection of serum antibody titer change of after adsorption sera,According to the best dilution of sera,the library was screened with in situ immunochemistry,to search the possible clones. However,no positive clones,which is pending further study.