Development Of Recombinant Overlapping Peptide Based On ESAT6 And CFP10 In Bovine Tuberculosis Diagnosis Reagent

Bovine Tuberculosis(b TB)is a zoonotic disease with long latent period,high infection rate,delayed onset,difficulty in treatment and lack of vaccine."Quarantine and culling" is the current prevention and control method of b TB.Therefore,developing an accurate,rapid,cheap and easy diagnostic method for b TB is a technical requirement for controlling the disease at home and abroad.At present,the quarantine method of b TB is intradermal test depending on tuberculin(PPD).However,due to the complex composition of PPD,it cannot distinguish the pathogenic mycobacterium tuberculosis from other mycobacteria and BCG,so sometimes the results are not accurate,which can cause more infection in human and animals.The current studies mainly improve the diagnostic accuracy of b TB by looking for specific antigens of M.bovis,including single ESAT6 and CFP10.The research problem is how to solve the false negative due to the low detection sensitivity and immune response stimulated by ESAT6 and CFP10.It is assumed that the two proteins(ESAT6 and CFP10)are designed to be recombinant overlapping peptide(ROP-TB)with more antigen sites exposed,which can improve the accuracy of b TB detection.To test the hypothesis,this study was conducted systematically by constructing plasmid,E.coli prokaryotic expression purification and identification,ROP-TB digestion by Cathepsin S in vitro and ROP-TB antigen presentation,challenging experiment in vivo in mice,guinea pig skin test and cow skin test.The results showed that the purity of ROP-TB derived from two Mycobacterium tuberculosis specific antigens(ESAT-6 and CFP10)could reach 100%,which was combined and made by prokaryotic expression.It can be cleavaged by Cathepsin S derived from lysosomal in vitro,ROP-TB with more antigen sites presented to T cells can stimulate stronger immune response than wild-type proteins(ESAT6 and CFP10).The results of tuberculosis models in vivo showed that specific T cell responses could be stimulated by ROP-TB in mice infected with M.tuberculosis.The results of skin test in guinea pigs infected with M.tuberculosis found that ROP-TB can stimulate stronger delayed-type hypersensitivity(DTH)responses compared with ESAT6/CFP10 and PPD(P<0.01).The statistical results of b TB skin test showed that all healthy cattles(n=40)were negative detected by PPD and ROP-TB,7 positive and 5 negative in suspected cattles(n=12)diagnosed by PPD and ROP-TB respectively.If we used PPD detection results as the standard,the sensitivity and specificity of ROP-TB were 85.7% and 97.7%respectively.In conclusion,the ROP-TB which is designed by protein recombinant overlapping and derived from two Mycobacterium tuberculosis specific antigens(ESAT-6,CFP10)can stimulate stronger response with more antigen sites presented to T cells.The spleen can be stimulated to release IFN-γ and the skin was stimulated to produce stronger response in animals.Therefore,it is proved that ROP-TB is an optimized skin test diagnostic reagent for b TB,which not only improves the specificity,but also has high sensitivity,and has a potential application prospect.