Cloning and Expression of virulent proteins CFP-10 and ESAT-6 of Mycobacterium bovis AN5 for diagnosis of bovine tuberculosis

Abstract

Aims and objects: Bovine tuberculosis caused by Mycobacterium bovis is an important disease that has negative effects in public health and economic losses. Traditional controlling program in cattle is based on test and slaughter strategy, but false positive is disadvantage of tuberculin skin test. To overcome the limitation, there is a need of using proteins with high specificity. The objective of this study was to clone and express of virulent protein CFP-10 and ESAT-6 of Mycobacterium bovis AN5 for diagnosis of bovine tuberculosis. Methods: Briefly, full-length genes of cfp-10 and esat-6 were amplified by PCR technique. In parallel pET23a (+) and PCR product has been double digested by EcoRI and HindIII. After Ligation, transformation was done into competent E.coli DH5α. Then the cloned vector was transformed into E.coli BL21. Induction was performed by isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed proteins almost entirely in the inclusion body form. To dissolution of expressed protein, urea 8M was used. Purification of recombinant proteins was done by Nickle-Resin. Urea eliminated by decreasing gradient. Delayed hyper sensitivity of two proteins beside bovine tuberculin, avian tuberculin and human tuberculin studied in guinea-pig model. Results: Protein expression was confirmed by Western Blotting (WB) using 6X His-tag antibodies. The results showed that CFP-10 and ESAT-6 molecules has been successfully cloned and expressed in prokaryotic system. Results showed that CFP-10 has satisfactory relative potency but it is not in range about ESAT-6. Conclusion: The recombinant CFP-10 can play a significant role in reducing the false positive in diagnosis of bovine tuberculosis by improving the specificity of the tuberculin skin test