Prokaryotic Expression Of Discrepant Genes Of Deer Tuberculosis Epidemic Strains And BCG And Establishment Of Indirect ELISA For Detection Of Antibody

Tuberculosis (TB) in deer mainly caused by Mycobacterium tuberculosis andMycobacterium bovis is a chronic and debilitating bacterial zoonotic disease of worldwidedistribution which makes a seriors threat to the healthy development of deer industry.Vaccination with Bacille Calmette–Gue′rin (BCG) is the approved attenuated live vaccine forprevention of TB, however the level of protection against TB provided by BCG vaccinationlacking of major protective antigen has been highly variable in different controlled trials aroundthe world. Animals vaccinated BCG would produce seriors interference with intradermaltuberculin skin test, therefore the skin test could not distinguish with the vaccinated deer andthe naturally infected deer.This study searched the detection method which could identify and diagnose thevaccinated deer and the naturally infected deer, thus established indirect enzyme-linkedimmunosorbent assay for detecting deer tuberculosis antibody. The main contents included thefollowing three aspects:(1) Clone of the discrepant genes and fusion genes of deer tuberculosis epidemic strainsand BCGFive discrepant genes Rv3872, Rv3873, Rv3874, Rv3875, Rv3878of RD1by specificprimers and two fusion genes Rv3874-Rv3875, Rv3872-Rv3874-Rv3875by Gene-SOE (genesplicing by over lap extension) method were amplified from the constructed gene library of deertuberculosis epidemic strain and BCG strain using the genomic DNA of deer tuberculosisepidemic strain which was isolated and identified in our laboratory as template. The sevenpurpose genes were inserted initially into pMD18-T simple vector respectively and the resultshowed that the seven target fragments were all imported into the cloned vector successfully.(2) Expression of the discrepant genes and fusion genes and analysis of their antigenicityThen these target fragments were sub-cloned into the expression vector pGEX-4T-1to gainprokaryotic expression. Constructed recombinant plasmids were induced with IPTG and thetarget proteins were purified by GST tag affinity chromatography purification system. Theresult showed that the purified proteins could interact with the positive serum of deertuberculosis with excellent antigenicity through SDS-PAGE and Western blot analysis.(3) Establishment of indirect ELISA detection of deer tuberculosis to identify the vaccinated deer and the naturally infected deerSeven ELISA detection methods were constructed respectively in the purified proteins asthe target diagnosis antigens. Besides, the optimized reaction conditions which includedconcentration of antigen, blocking liquid, serum, IGg-HRP and substrate, established the sevendetection methods of indirect ELISA. Then critical values of positive and negative serums wereconfirmed. Through the sensitivity, specificity and repitation tests, the result showed that theseven detection methods of indirect ELISA for each antigen had higher sensitivity, specificityand repeatability.The study cloned and expressed the discrepant genes and fusion genes of deer tuberculosisepidemic strains and BCG, and provided several practicable antigens for establishment of thediagnose method to identify the vaccinated deer and the naturally infected deer. Furthermore thestudy laid a foundation for applying these proteins in serological diagnosis of deer tuberculosisin the future.