The Expression Of Candidate Antigens Of Lawsonia Intracellularis And The Application In Indirect ELISA Method

Lawsonia intracelluaris(LI),a member of Family Desulfovibriom,is an abligate intracellular pathogen.It is microaerobic,Gram-negative and acid-fast-positive staining and the form is curved,rod-shaped or comma-shaped,with the length of about 1.25 ?m-1.75 ?m and the width of about 0.25?m-0.43?m.LI is one of the pathogens that cause porcine intestinal diseases.It is mainly parasitized on the epithelial cells of the porcine ileum,causing porcine proliferative ileitis(also known as proliferative enteropathy),leading to slow growth of pigs and bringing to the pig industry dramatically economic loss.Since it was first discovered in pig intestinal adenomatous lesions in 1931,it has been confirmed that the bacteria can naturally infect horses,dogs,hamsters,rabbits,foxes,cats,sheep,deer,ostriches,chickens and other animals.LI infections occur in pig-raising countries in the world,such as the United States,Canada,Australia,Europe,Brazil,Japan,South Korea,China,etc.The methods currently used to detect LI include polymerization and plum chain reaction(PCR),indirect immunofluorescence test(IFA),immunoperoxidase monolayer assay(IMPA),enzyme-linked immunosorbent test(ELISA).Up to date,poor investigations for this economically important disease to pig production were carried out in China,and no commercial detection kits could be used.In this study,we developed an indirect ELISA method using a predicted flagellate protein for the epidemiological monitoring and clinical detection.In this study,according to the predicted proteins reported in the literature,Lawsonia intracellularis flagella-related proteins(LI0641,LI0570,LI0710),opp A protein LI0169,outer membrane protein LI0841,effector protein LI1035 and membrane transporter LI1153 were selected as candidate proteins.The recombinant plasmids p ET32?-LI0641,p ET32a-LI0570,p ET32?-LI0710,p ET32?-LI0841,p ET32?-LI0169,p ET32?-LI1153,p ET32?-LI1035 were successfully constructed,transformed into E.coli BL21(DE3)competent cells,expressed by IPTG and purified by a nickel column,SDS-PAGE electrophoresis identified the candidate protein successfully expressed and the purification effect was good,later the recombinant protein was precipitated and purified with anti-E.coli serum,and then the concentration of each recombinant protein was determined to be 2.41 mg/ml and 2.57 mg/ ml,3.24mg/ml,2.04 mg/ml,2.32 mg/ml,2.67 mg/ml,1.52 mg/ml.The seven recombinant proteins were first verified by Western blot for their reactogenicity.Among them,r LI0570,r LI0710,r LI0841,and r LI1035 were reactogenic,and then the immunogenicity of these four proteins was verified by IFA,and the results showed that they all have immunogenicity.Subsequent bioinformatics analysis of r LI0841 protein does not have antigenic epitope crossover with other pathogens and has good reactogenicity.The r LI0841 protein was selected as the coating antigen to create an indirect ELISA method for detecting LI.Through the basic optimization of indirect ELISA,the best conditions are determined;afterwards,it is specifically verified that the recombinant antigen has no cross-reactivity with other pathogens(Actinobacteria,Haemophilus parasuis,Coronavirus,Transmissible gastroenteritis virus,Escherichia coli,Salmonella,Clostridium perfringens,Streptococcus,Mycoplasma),Showing high specificity;sensitivity test results show that the serum dilution is 1:800 and 1:1600 respectively positive,showing high sensitivity.The created indirect ELISA was used for repeatability experiments.The intra-assay coefficient of variation was less than 5.2%,and the inter-assay coefficient of variation was less than 5%,which showed good repeatability.The positive rate of mouse seropositive detection by the established indirect ELISA method was 0;the positive rate of the two pig farms in Gansu and Jiangsu provinces were 15% and 22%,respectively.In summary,this study successfully developed an indirect ELISA method for detecting antibodies against Lawsonia intracellularis,which provides a powerful tool for seroepidemiological investigation of Lawsonia intracellularis and clinical diagnosis of porcine proliferative ileitis..