Expression Of LS1087 And LS1136 Proteins Of Lawsonia Intracellularis And Preparation And Application Of Their Monoclonal Antibodies

Porcine ileitis(porcine proliferative enteropathy,PPE)is a kind of intestinal tract caused by Lawsonia intracellularis with colon and cecal mucosa at the proximal end of the ileum and cecum disease.Lawsonia intracellularis mainly damages the intestines of pigs and seriously affects the ratio of meat to meat in pigs.The genome of Lawsonia intracellularis has more than 1.7 million base pairs and has 1,324 protein coding regions.Although some proteins such as LsaA protein,LatA protein and LhlyA protein have been reported,many proteins are still unknown.The LS1087 protein is a putative membrane protein of Lawsonia intracellularis,and the LS1136 protein is a hypothetical protein of Lawsonia intracellularis.In this study,the DNA was extracted from ileitis-positive disease material,and LS1087 and LS1136 proteins were obtained by PCR,plasmid construction and prokaryotic expression.Two kind of monoclonal antibodies against these two proteins were prepared by hybridoma technology,which laid the foundation for further research of Lawsonia intracellularis.1.Prokaryotic expression of LS1087 protein and LS1136 proteinPrimers were designed according to the full-length sequence of Lawsonia intracellularis(Accession number: AM180252.1)in GenBank.DNA was extracted from ileitis-positive disease material,and the LS1087 and LS1136 gene fragments were obtained by PCR.The LS1087 gene and the LS1136 gene were cloned into the prokaryotic expression vectors pET-32 a and pGEX-4T-1,and the recombinant expression vectors pET-32a-LS1087,pGEX-4T-1-LS1087,pET-32a-LS1136 and pGEX-4T-1-LS1136 were constructed.The recombinant expression vectors were transformed into the expression strain,and recombinant proteins LS1087-His,LS1087-Gst,LS1136-His,and LS1136-Gst were expressed.The results of SDS-PAGE showed that the fusion proteins were mainly in the form of inclusion bodies.The fusion proteins LS1087-His,LS1087-Gst,LS1136-His,and LS1136-Gst were 38 kDa,44 kDa,55 kDa and 64 kDa,respectively.LS1087-His and LS1136-His proteins were purified using a nickel column,and LS1087-Gst and LS1136-Gst proteins were purified using DTT,DOC(sodium deoxycholate)and SKL.Results shows that LS1087-His and LS1136-His protein had the better purification effects than LS1087-Gst and LS1136-Gst protein.2.Preparation of monoclonal antibodies against LS1087 protein and LS1136 protein The LS1087-Gst and LS1136-Gst protein were mixed and it was used as an immunizing antigen to immunize BALB/c mice,and LS1087-His and LS1136-His proteins were mixed as detection antigens.The mouse which has highest antibody level were determined by ELISA square matrix titration and indirect ELISA.5 hybridoma cells stably secretingmonoclonal antibodies were successfully screened conducted by hybridoma cells,named as 7E9,9D8,15H10,16A1 and 16G10.The results of ELISA and Western blotting showed that 7E9 and 9D8 were resistant to LS1087 protein;15H10,16A1 and 16G10 were resistant to LS1136 protein.The results of specificity experiment showed that there was no obvious cross-reaction between the five monoclonal antibodies and the other nine bacterial supercracks.Immunohistochemistry showed that 5 monoclonal antibodies could specifically bind to Lawsonia intracellularis.At present,there is no report on the preparation of monoclonal antibodies against the intracellular Lawsonia protein in China.In this study,monoclonal antibodies against the Lawsonia intracellularis protein were prepared,and 5 strains capable of specifically binding to the intracellular Lawsonia were successfully obtained.These 5 monoclonal antibodies can be used as the tools for the isolation or detection of Lawsonia,laying the foundation for further research.