Cloning And Expression Of Surface Protein Of Lawsonia Intracellularis And Establishment Of ELISA Method For Detection Of Antibody

Porcine proliferative enteropathy(PPE)is an infectious intestinal disease mainly characterized by the proliferation of intestinal epithelial cells and thickening of the intestinal mucosa caused by Lawsonia intracellularis(LI)in pigs.The disease is one of the common diseases in intensive farms,which is widely prevalent in major pig-raising countries in the world.In addition to the occasional acute death of large and mediumsized pigs,the main harm to the pig industry is the widespread chronic infection of LI in the pig population,which seriously affects the feed return rate and causes significant economic losses to the global aquaculture industry.Since July 2020,China has completely banned antibiotics in feed,and the harm caused by LI infection is likely to increase.The purpose of this study is to screen the effective LI surface proteins by cloning and expression technology on the basis of bioinformatics analysis and to establish an indirect ELISA antibody detection method for LI,which could provide a basis for the epidemiological investigation of LI and the development of antibody detection kits in China.1.Bioinformatics analysis of the complete LI gene sequenceIn this study,CELLO(http://cello.life.nctu.edu.tw/)and PSOTRb(http://psort1.hgc.jp/form.html)were used to predict the subcellular localization of all proteins encoded by open reading frames of LI strain(Gen Bank number : NC＿008011.1 and CP004029.1).A total of 34 surface proteins of LI were predicted(A cytoplasmic protein(law-7)was mistakenly counted as a surface protein in the statistical prediction process,and the protein was cloned and expressed,so it was collectively called 34 surface proteins in this paper).TMHMM and Signal P in the website(http://www.cbs.dtu.dk/services/)were used to predict the transmembrane region and signal peptide coding region of these proteins.2.Cloning,expression and purification of 34 LI surface proteinsThe gene sequences of 34 proteins after removing transmembrane and signal peptide coding regions were cloned into prokaryotic expression vector p ET-28a(+)(Among the 34 proteins,the gene fragments of 3 proteins were too long to be truncated into 2 genes fragments according to their functional domains predicted by BLAST in NCBI.Then the predicted 34 surface proteins were divided into 37 gene fragments for cloning and expression)and transformed into E.coli for expression.A total of 28 recombinant proteins were successfully expressed,of which 7 proteins were expressed in soluble form and 21 proteins were expressed in inclusion body form.The 28 recombinant proteins were purified by nickel affinity chromatography(4 of them were insoluble in urea and only washed by inclusion bodies washing solution).3.Establishment and application of LI indirect ELISA antibody detection methodA flagellar hook-basal body complex protein flg E was screened from 28 expressed LI surface proteins by preliminary pre-test,which had obvious reaction signal with LI positive serum,high positive detection rate and low negative serum background.It was used as coating antigen to optimize the reaction parameters by matrix titration,and the ELISA method(flg E-ELISA)for detecting LI antibody was preliminarily established.Specific tests showed that flg E-ELISA method had no any cross reactions with other positive sera,such as the positive sera of CSFV,PRV,PRRSV,PCV2,FMDV,Mhp and APP.It can detect the LI positive serum with the maximum dilution of 1600 and the coefficients of variations in both inter-and intra-assay were less than 10%.Compared with the commercial kit(SVANOVIR?L.intracellularis/Ileitis-Ab)of Swedish Svanova company,the coincidence rate of negative serum and sows serum was higher,but flg E-ELISA method was more sensitive than the commercial kit in detecting the serum of early LI infection.Using flg E-ELISA method to detect different types of pig serum,the results showed that the infection rate of sows and fat pigs was up to 100 %.After the decrease of maternal antibody,the antibody level and positive rate were low at 30 days of age,and increased with the increase from the age of 50 to 70 days to the finishing pig stage.Under separate closed feeding conditions,it took a short time for pigs to infect LI from the beginning to the whole group.These results showed that the indirect ELISA antibody detection method established in this study can accurately reflect the antibody level and antibody positive rate of LI infected pigs,with good specificity,sensitivity and repeatability,which can be preliminarily used for clinical epidemiological investigation of LI.