Establishment And Primary Verification Of Indirect ELISA Detection For Deer Tuberculosis To Identify The Wild Infection And BCG Immunization

Deer tuberculosis is mainly caused by Mycobacteriumbovis, a chronic, debilitating zoons. The course of the disease is distributed into whole world. People’s health and the development of the animal husbandry is threaterned seriously. With the rising number of the deer farming, the breeder by contact with sick animals infected with TB cases is rising year by year. Therefore, control the spread of the deer tuberculosis not only helps to reduce the economic loss of stag breeding, but also reduces the risk of the breeder’s breeding.At present, BCG vaccine was mainly used the to control TB in our country. BCG was inoculated into the deers and could reduce the degree of the TB. But the result is not stable. BCG was inoculated into the animals for a long time. The tuberculin skin test results were false positive, which seriously influenced the tlts using SDS-PAGE.RD1 and RD5 all exist in the mycobacterium tuberculosis, missing in the BCG vaccine. Sources confirmed that the RD5 area Rv3117 gene is able to distinguish between animal tuberculosis natural infection or BCG vaccine immunization, and the sensitivity and specificity were higher than ESAT-6 protein. This research cloning, expression and purification of the Rv3117 protein, induced the laboratory preservation zone RD1 gene recombinant expression plasmid and purified to obtain Rv3117, Rv3872, Rv3873, Rv3874, Rv3875, Rv3878, protein and Rv3872 Rv3874-3875-3875-3874. Using ELISA method comprehensive evaluation of the protein as a deer TB ELISA detection method of the sensitivity and specificity of candidate antigens, select the optimal protein antigen Rv3872-3874-3874, set up a kind of deer to identify wild strain of tuberculosis infection with BCG vaccine immunization method, thus reducing the deer TB ELISA detection of false positives.This research mainly involved the following contents.(1) Rv3117 was cloned and expression vector was established.Specific primer was designed as a Standard of mycobacterium tuberculosis bacterium H37 Rv template. Mycobacterium tuberculosis was only existed in the amplification, but lost in the BCG Rv3117 RD5 area specific genes. Rv3117 and gene cloning vector pMD18-T were connected. After it has been identification results showed that the Rv3117 gene was cloned to the cloning vector. Rv3117 gene was connectted to expression vector pGEX-4T-1 and was transformed into E. coli BL21(DE3) cells, screening positive clones.(2) The expression and purification of differences gene of epidemic strains and BCGTo early preparation of pGEX-4T-1-Rv3117 strains were induced and was tested if it has antigenicity of mycobacterium tuberculosis with Western blot method. A large number of induced Rv3117 protein and mycobacterium tuberculosis preserved in our laboratory RD1 region strains of specific genes were expressed. They were purified using the method of affinity chromatography and were analyzed resuuberculin skin test results.(3)Identification of wild deer tuberculosis strains and BCG optimal antigen detection ELISA Screening.Because this method does not have controlled trials, I prepared rabbit artificial infected with TB and non-tuberculosis mycobacterium tuberculosis bacteria and vice and BCG positive serum. The specific protein was regarded as envelope antigen, to establish indirect ELISA method respectively, comparing the envelope antigen to establish indirect ELISA method repeatability, sensitivity and specificity. Verified by Rv3872-74-75 protein has high repeatability, sensitivity and specificity, can distinguish between the natural infection and the BCG vaccine immunization, which could prepare the way for later establish deer TB diagnosis method.(4) Establishment and primary application of indirect ELISA detection for deer tuberculosis to identify the wild infection and BCG immunizationThe method of molecular biology was used in the study. The mycobacterium tuberculosis specific proteins was cloned and induced to express. Various protein as the nature of the envelope antigen was compared by ELISA method. And finally established Rv3872-74-75 protein as envelope antigen to establish an indirect ELISA detection deer TB which lay the ground for deer serological diagnosis of tuberculosis.