Prokaryotic Expression Of Goat Corynebacterium Pseudotuberculosis PLD And Establishment And Application Of Indirect ELISA Method

Caseous lymphadenitis of goat,is an infectious disease caused by Corynebacterium pseudotuberculosis,the characteristics of which are the formation of abscesses in the lymph nodes and visceral organs.The host range infected by this pathogen is extremely widespread,apart from sheep and goats that are susceptible to the infection,other ruminants and humans can be infected.Infected goats carry a large number of pathogenic bacteria.Improper handling can cause harm to human dietary hygiene and physical health,posing a potential threat to public safety and health.Therefore,rapid and accurate detection of the infection of the pathogen not only helps to control the occurrence of C.pseud-otuberculosis disease in goat breeding,but also provides guarantee for public health safety.At present,the diagnostic methods of infection with C.pseudotuberculosis including isolation and culture identification of pathogens,PCR detection and serological diagnosis,etc.Among these detection methods,the first and second methods are usually diagnosed after the clinical symptoms appear,clinically,early infection or visceral infection may be missed diagnosis.Therefore,it is of great value to diagnose the early infection of the pathogen by serology method.Phospholipase D(PLD)is a major virulence determinant of C.pseudotuberculosis,which is crucial to the spread of the pathogen.It is often used as the research target of vaccine and diagnostic methods.The aim of this study was to express the recombinant PLD protein(r PLD)of C.pseudotuberculosis,and establish an ELISA detection method based on the PLD antibody of C.pseudotuberculosis infection.On this basis,epidemiological investigation and analysis were carried out on the serum of some sheep infected by C.pseudotuberculosis in Chongqing,providing scientific basis for the prevention and control of the disease.The main research contents and results are as follows:1.The prokaryotic expression of phospholipase D of C.pseudotuberculosis,bio-logical activity test and polyclonal antibody preparationFirstly,a prime for amplifing the full-length sequence of PLD gene was designed,and then the cloning plasmid p MD19-T-PLD was amplified and constructed.After double enzyme digested and connected with p Cold-TF to construct recombinant expression vector p Cold-PLD,it was transferred into host strain BL21 to induce expression.The expressed r PLD protein was detected by SDS-PAGE and purified,and the bioactivity was detected by synergic hemolysis with the culture supernatant of Rhodococcus equi.Immunized rabbits with r PLD protein to prepare anti-PLD hyperimmune serum,then AGAR diffusion test and Western-blot identification were carried out.The results showed that the molecular weight of the PLD fusion protein induced by SDS-PAGE was 85.8 k Da(p Cold-TF 52k Da+PLD33.8k Da),purified r PLD protein can produce synergistic hem-olysis reaction with Rhodococcus equi supernatant.The results of the AGAR diffusion experiment showed that r PLD protein produced precipitation lines with the rabbit anti-PLD hyperimmune serum and the positive serum of goats infected by C.pseudotuberculosis,with the titer 1:32 and1:16 respectively.Western-blot results showed that the r PLD protein could produce a single hybridization band with the positive serum of goats infected by C.pseudotuberculosis.In conclusion,it was shown that the r PLD protein of C.pseudotuberculosis with good bioactivity was successfully expressed,it could have specific reaction with rabbit anti-PLD serum and positive serum of goats infected by C.pseudotuberculosis.2.Establishment of an indirect ELISA method for detecting PLD antibodies of C.pseudotuberculosisThe r PLD protein was used as coating antigen.The conditions of the indirect ELISA,such as the concentration of the coating antigen,the coating conditions,the dilution of the serum,the time of the enzyme-labeled secondary antibody and other conditions were scree-ned to determine the critical value of the positive and negative serum of C.pseudotbe--rculosis,then the sensitivity,specificity,repeatability were tested and evaluated to establish ELISA method for serological detection of C.pseudotuberculosis.The results showed that the antigen coating concentration was 8?g/m L,the positive serum dilution was 1:800,and the optimai condition of coating at 4?for 12 h.After blocked at 37?for90 min with 5%skimmed milk powder and added the positive serum incubated for 30 min,then the enzyme-labeled secondary antibody was diluted to 1:8000 and incubated at 37?for 30 min,taken it out and a chomogenic substrate was added to react 15 min.According to definition of 40 negative sera,the critical value of the negative and positive serum of C.pseudotuberculosis was 0.193.When the positive serum was diluted to 1:51200,the OD₄₅₀ value detected by ELISA was still higher than the critical value.The established ELISA method was no cross-react with the positive sera of goats infected by Cryptococcus pyogenes,Staphylococcus aureus and Acinetobacter baumannii,the coefficients variation range of repeated tests within and between panels was less than 10%.The results showed that the established indirect ELISA method for detection of C.pseudotuberculosis PLD antibody had good sensitivity,specificity and repeatability.3.Serum epidemiological investigation of C.pseudotuberculosis infection in goat farms in some areas of ChongqingThe established ELISA method was applied to investigate the serum epidemiology of C.pseudotuberculosis infection in some goat farms in 11 counties and districts of Chongqing.The results showed that the overall positive rate of C.pseudotuberculosis infection in Chongqing was 47.62%(95%CI=40.30-55.00%).Of all the tested samples,the highest positive rate of C.pseudotuberculosis infection in Qianjiang was 91.67%(95%CI=61.50-99.80%),and the second was Youyang with 85%(95%CI=62.10-96.80%),In addition to the fact that C.pseudotuberculosis was not detected in Rongchang serum samples(0%,95%CI=0-30.80%),the positive rates of C.pseudotuberculosis infection in other districts and counties were 5.00-60.00%.The results indicated that most districts and counties in Chongqing were infected with C.pseudotuberculosis,and the infection rate varied greatly among districts and counties.In summary,the r PLD protein of C.pseudotuberculosis with good bioactivity was successfully obtained,indirect ELISA method for detecting PLD antibodies of C.pseudotuberculosis was established in this study,providing technical support for serum epidemiological investigation and immune antibody level monitoring of goats infected with the pathogen.Using established ELISA method for dection,it was found that the infection rate of C.pseudotuberculosis in goats in some counties in Chongqing was relatively high with 47.63%which should be paid attention to by farmers?animal husbandry and vete-rinary workers.