Authors : Shuqing Liu, Xintao Gao, Pingjun Li, Hong Jia, Ting Xin, Shaohua Hou, Xiaoyu Guo, Weifeng Yuan, Gaimei Zhang, Ming Li, Jing Wu and Hongfei Zhu
Abstract: Bovine tuberculosis caused mainly by Mycobacterium bovis is a serious zoonosis in the world that can infect cattle and humans as well. Prevention and eradication of this disease require effective and sensitive diagnosis method. While the traditional tuberculin skin test and IFN-γ release assay stimuli as PPD-B may share common antigens with Mycobacterium avium which could cause false positive results. To overcome this problem, studies are focus on screening more specific proteins to replace PPD for stimuli recently. Previous research showed that TB10.4 could activate T cell stimulation therefore, researchers optimized the expression of TB10.4 by comparing three different recombinant proteins and using as supplementary antigen stimuli was detected by in diagnosis of bovine tuberculosis. The pET-32a (+) plasmid and pET-28a (+) plasmid were chosen which are different in the solubility of the recombinant protein. After transformation, expression and purification of the recombinants, the recombinant proteins rTB10.4-1 and rTB10.4-3 were obtained successfully. In order to analysis the importance of the C terminal for the activity of TB10.4, the initiation and termination codon were also introduced into the N and C terminal of TB10.4 gene and the rTB10.4-2 lack of His tag protein in the C terminal was obtained. The result of SDS-PAGE showed that rTB10.4-1, TB10.4-2 and rTB10.4-3 were 30, 30 and 12 ku, respectively which were just the same size as expected. The biological activities of these recombinant-proteins were compared by IFN-γ release assay, real-time PCR and Western blot and the results showed that all of the proteins had good T cell activity and B cell activity. In comparison, rTB10.4-1 possessed strongest activity while rTB10.4-3 was the lowest one as the inclusion body. The skin test on guinea pigs showed that the DTH in M. bovis-infected group could be detected intensively by using mixture of recombinant proteins rTB10.4-1 and rCE in skin test compared to control group. In summary, the recombinant protein of TB10.4 could be obtained successfully and be used as antigen stimuli in the skin test which would lay the foundation for manufacture of the diagnosis kit for bovine tuberculosis with characteristics of high specificity, sensitivity and reproducibility.
Shuqing Liu, Xintao Gao, Pingjun Li, Hong Jia, Ting Xin, Shaohua Hou, Xiaoyu Guo, Weifeng Yuan, Gaimei Zhang, Ming Li, Jing Wu and Hongfei Zhu, 2014. Expression of Recombinant TB10.4 of Mycobacterium bovis and the Preliminary Study of its Potential in Diagnosis of Bovine Tuberculosis. Journal of Animal and Veterinary Advances, 13: 549-557.