Abstract：This study was to express the fusion protein of the culture filter protein 10 (CFP10) and earlier secreted antigen target 6 (ESAT6) of Mycobacterium tuberculosis in the yeast Pichia pastoris system and to evaluate its potential in bovine tuberculosis (bTB) detection. The fusion fragment cfp10-esat6 was amplified by PCR and constructed into the recombinant plasmid pPICZαA-(cfp10-esat6), which was transformed into the yeast P. pastoris GS115 by high voltage electroporation. The positive recombinant yeast was induced for 3 d in the culture medium containing 1% methanol. The fusion protein of CFP10-ESAT6 (eCE) was analyzed by SDS-PAGE and then purified by nickel ions affinity chromatography column which targets the histidine (His) tag. Immunological reactivity of eCE was also appraised by Western blot. Furthermore, the application potential of eCE in bTB detection was evaluated via using it as stimulator in bovine peripheral blood interferon gamma release test in vitro (IFN-γ release test). The results showed that eCE successfully expressed in the recombinant yeast and secreted into the culture supernatant after induced by methanol. Results of Western blot demonstrated that only one specific band appeared when the fusion protein reacted with monoclonal antibodies of CFP10, ESAT6, His tag and c-Myc proteins, respectively, indicating that eCE proved an efficacious immunological reactivity. In the IFN-γ release test for 165 cow blood samples, eCE had a positive coincidence rate of 82.3% as well as a negative coincidence rate of 78.8% compared with purified protein derivative (PPD) used as stimulator in bTB detection. In summary, eCE expressed successfully in the yeast P. pastoris system and exhibited an excellent biological activity. It can be used as stimulator like PPD in IFN-γ release test for the diagnosis of bTB, as well as improving the sensitivity and specificity of the test.
Benabdesselem C, Fathallah D M, Huard R C, Zhu H, Jarboui M A, Dellagi K, Ho J L and Barbouche R M, 2006. Enhanced patient serum immunoreactivity to recombinant Mycobacterium tuberculosis CFP32 produced in the yeast Pichia pastoris compared to Escherichia coli and its potential for serodiagnosis of tuberculosis. Journal of clinical microbiology, 44(9):3086~3093Cregg J M, Vedvickand T S and Raschke W C, 1993. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology, 11(8):905~910 Gormley E, Doyle M B, Fitzsimons T, McGill K and Collins J D, 2006 . Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon (Bovigam) assay. Veterinary microbiology, 112(2-4):171~179Lewis K N, Liao R, Guinn K M, Hickey M J, Smith S, Behr M A and Sherman D R, 2003. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guérin attenuation. The Journal of infectious diseases, 187:117~123Macauley-Patrick S, Fazenda M L, McNeil B and Harvey L M, 2005.Heterologous protein pr oduction using the Pichia pastoris expression system. Yeast, 22:249~270MENG C, WAN T, XU Z Z, SHAN F, XIE X L, SHEN J S, FAN F, CHEN X and JIAO X A, 2014. Fusion Expression of Mycobacterium tuberculosis ESAT6 and CFP10 protein and its cellular immunoproperties. Acta Veterinaria et Zootechnica Sinica, 2014, 45(5):789~794 (孟闯, 万婷, 徐正中, 单 法, 解晓莉, 申峻松, 范 峰, 陈 祥, 焦新安, 2014. 结核分枝杆菌ESAT6-CFP10蛋白的融合表达及其细胞免疫学特性. 畜牧兽医学报, 45(5):789~794)Palmer M V and Waters W R, 2006. Advances in bovine tuberculosis diagnosis and pathogenesis: what policy makers need to know. Veterinary microbiology, 112(2-4):181~190Renshaw P S, Lightbody K L, Veverka V, Muskett, F W, Kelly G, Frenkiel T A, Gordon S V, Hewinson R G, Burke B, Norman J, Williamson R A and Carr M D, 2005. Structure and function of the complex formed by the tuberculosis virulence factors CFP10 and ESAT6. The EMBO journal, 24(14):2491~2498Sambrook J and Russell D W, eds, 2001. Molecular Cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, New YorkSHEN J S, 2008. Prokaryotic expression of Mycobacterium tuberculosis lhp gene and development of monoclonal antibodies against the product. Thesis for M.S., YangZhou University, Supervisor: JIAO X A, pp.23~30 (申俊松, 2008. 结核分枝杆菌lhp基因的原核表达及其产物的单克隆抗体研制. 硕士学位文, 扬州大学, 导师: 焦新安)Timothy C R, Marisa M, Kathleen S M, Brodine S K and Strathdee S A, 2008. Tuberculosis from Mycobacterium bovis in Binational Communities, United States. Emerging infectious diseases, 14(6): 909~916Weinrich O A, Van pinxteren L A, Meng okkels L, Birk Rasmussen, P and Andersen P, 2001. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT6. Infection and immunity, 69(5):2773~2778World Health Organization, 2013. Global tuberculosis control 2012. GenevaWorld Organization for Animal Health (OIE), 2009. Manual of diagnostic tests and vaccines for terrestrial animals. Paris
