Prokaryotic Expression The Candidate Antigen Protein Of Porcine Lawssonia Intracellularis,Establishment Of An Inderect Elisa For Detection Of Antibody Against Lawsonia Intracellularis And The Preliminary Application

We have successfully amplified the gene sequences of four candidate antigen proteins from Lawsonia intracellularis positive porcine ileal mucosal DNA by PCR or Nested-PCR. The four candidate antigen proteins are outer membrane protein1022, outer membrane protein0902, outer membrane protein1024and surface lipoprotein0235. The PCR products are respectively cloned to pMD-18T, and then subcloned to prokaryotic expression vector pET-32a(+). The four recombinant expression plasmids pET32a-OMP1022, pET32a-OMP0902, pET32a-OMP1024and pET32a-SLP0235were identified by double enzyme and sequencing.The four recombinant expression plasmids were transfected into BL-21(DE3) and induced by IPTG. The recombinant protein1022and0902were expressed, but recombinant protein1024and0235have not been expressed. We then segmented amplified OMP1024by two fragments, named OMP1024a and OMP1024b. The recombinant expression plasmids were successfully structured and transfected into BL-21(DE3). The recombinant protein1024a and1024b were expressed induced by IPTG. The recombinant protein1022,0902,1024a and1024b could specifically react with Lawsonia Intracellularis positive porcine serum. The recombinant proteins were purified by Ni2+chelating affinity chromatography. The purified and renatured recombinant protein1022,0902,1024a and1024b all have good antigenicity identified by Western-blot. Among them,1024b have the best antigenicity.The recombinant protein1024b were used as the coating antigen to establish the indrect ELISA. We carried out a series of tests to determine the optimum reaction conditions, and the results are:The optimal concentration of coated antigen is0.5μg/mL, and coated for2h at37℃; The optimal dilution of serum is1:40, and the best reactive time is1h; The optimal dilution of second antibody is1:8000, and the best reactive time is30min; The color rendering time of TMB substrate is min at room temperature; The positive criterion of this ELISA method is OD450≥0.321and P/N≥2.1. The results of serum adsorption test, cross-reaction test and competitive inhibition test suggested the LI-1204b indirect ELISA is specific to Lawsonia Intracellularis.By application of the established LI-1204b indireet ELISA method for diagnosis,648porcine serum samples were tested which were collected from different parts of GuangXi Province. The sesults showed that436serum samples were positive, the postive rate was67.3%. The positive rates of serum samples collected from NanNing, LiuZhou, GuiLin, GuiGang and YuLin are52.7%, 81.1%,76.5%,68.0%and71.8%. The positive rates of piglets, suvery pigs, finishing pigs, sows and boars respectively are50%,30.8%,61.9%,92.4%and88.6%.