Establishment And Preliminary Application Of Indirect Elisa Method For Detection Of Corynebacterium Pseudotuberculosis Antibody Based On PLD Protein

Corynebacterium pseudotuberculosis(Cp)infection causes Caseous lymphadenitis(CLA),also known as goat pseudotuberculosis.Abscess and caseous necrosis appear in superficial lymph nodes,liver,lung and other organs of sheep and goats,which seriously restricts the healthy development of sheep breeding industry in China.At present,there is no effective treatment and prevention measures for this disease,and the immune effect of the existing vaccines at home and abroad is poor,which can not play a good role in protecting animals,and there is no good diagnostic method for this disease.Therefore,it is urgent to establish the detection method of Corynebacterium pseudotuberculosis antibody for clinical application,timely screening of infected sheep,to achieve the purpose of disease purification.In this study,the pld gene of Corynebacterium pseudotuberculosis was expressed in E.coli and the PLD protein was purified.Then an indirect ELISA method based on PLD protein of Corynebacterium pseudotuberculosis was established for the detection of antibodies,and the method was initially applied to clinical detection.The results are as follows:1.The pld gene(924 bp)and cp40 gene(1134 bp)of Corynebacterium pseudotuberculosis Shaanxi isolate were cloned successfully.The nucleotide sequence of pld gene shared 98%?100% homology with other Corynebacterium pseudotuberculosis pld genes,and the nucleotide sequence of cp40 gene shared 91%?99% homology with other Corynebacterium pseudotuberculosis cp40 genes.Phylogenetic analysis showed that pld gene of Corynebacterium pseudotuberculosis isolated from Shaanxi province was closely related to British strain NCTC4681,and cp40 gene was closely related to Brazilian strain C231.The deduced amino acid sequences of pld and cp40 have no signal peptide,no transmembrane domain,strong hydrophilicity and many surface antigens.2.The recombinant prokaryotic expression plasmids pet-28a-pld and pet-28a-cp40 were constructed to transform BL21.A large number of high purity proteins were purified by Ni column affinity chromatography.The two recombinant proteins were 33 ku PLD protein and41 ku CP40 protein respectively.Western blot analysis showed that the two recombinant proteins could be combined with the positive serum of Corynebacterium pseudotuberculosis and have certain reactivity.The purified PLD protein was used as the enveloping antigen to establish an indirect ELISA method for the detection of antibody against Corynebacterium pseudotuberculosis.Optimum antigen package quantity was 200 ng,the serum to be examined for 1: 200 dilution,the enzyme standard II antibody was diluted with 1:5 000,the time of action was 1h,and the time of TMB was 20 min.The specificity,sensitivity and repeatability were good,and the coincidence rate was high.3.Using PLD protein as coating antigen,the establishment of colloidal gold immunochromatographic assay for detection of Corynebacterium pseudotuberculosis antibody was studied.Among them,the sandwich method of PLD protein labeling,the indirect method based on G protein and Rabbit anti goat Ig G were tested respectively.At present,the three methods are not successful and need to be further explored.