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| Expression, Purification and Immunogenicity Analysis of Recombinant CFP10-ESAT-6-MPB64 Using the Baculovirus Expression System |
| XU Dan1,2,4, LIU Min1,2, KONG Ju3, LI Xiao-kun1,2, JIANG Chao1,2 |
1. Key Laboratory of Biotechnology Pharmaceutical Engineering of Zhejiang Province, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325035, China;
2. Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Changchun 130118, China;
3. Juye County Peoples Hospital, Heze 274900, China;
4. Jinan Kanghe Pharmaceutical Technology Co., Ltd. Jinan 250101, China |
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Abstract Objective: To express Mycobacterium tuberculosis protein CFP10-ESAT-6-MPB64 in baculovirus insect cell expression system, and identify its immunogenicity. Methods: The target gene CFP10-ESAT-6-MPB64 was connected to pFastBac vector, then the pFastBac-CFP10-ESAT-6-MPB64 plasmid which was harvested would transformed to DH10Bac competent, and the target gene was transposition into Bacmid by Tn7 transposase fragment, therefore Bacmid-CFP10-ESAT-6-MPB64 Shuttle vector was obtained. The shuttle vector was packaged by liposomes and transfected Sf9 cells to harvest P1-generation virus, then high titers of P4 generation virus was harvested by repeat transfected Sf9 cells three times. The target protein CFP10-ESAT-6-MPB64 was purified from the supernatant by Co affine chromatography, which were used to immunize Balb/c mice. Antibody changes in serum would be detected, and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γ secretion by CFP10-ESAT-6-MPB64 stimulated spleen cells by ELISA method. Result: CFP10-ESAT-6-MPB64 successfully expressed in insect cells. The purity of target protein is over 90% and yield up to 42mg/L after purification. Purified protein can effectively stimulate Balb/c mice to produce antibodies, increase the content of IFN-γ medium in mice spleen cells, and significantly promoting proliferation in spleen cells between 1~50μg/ml. Conclusion: CFP10-ESAT-6-MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system, that open a new avenue for tuberculosis vaccine production.
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Received: 06 May 2014
Published: 25 June 2014
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[1] http://www.who.int/tb/publications/global_report/gtbr12_executivesummary.pdf.
[2] Wright A, Zignol M, Deun A V. Epidemiology of antituberculosis drug resistance 2002-07: an updated analysis of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance. Lancet, 2009, 373: 1861-1873.
[3] Shah N S, Wright A, Bai G H. Worldwide emergence of extensively drug-resistant tuberculosis. Emerg Infect Dis, 2007,13: 380-387.
[4] Gandhi N R, Moll A, Sturm A W. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet, 2006,368:1575-1580.
[5] Harries A D, Zachariah R, Corbett E L. The HIV-associated tuberculosis epidemic-when will we act. Lancet, 2010,375:906-1919.
[6] Ghebreyesus T A, Kazatchkine M, Sidibé M. Tuberculosis and HIV: time for an intensified response. Lancet, 2010,375:1757-1758.
[7] Fine P E. Variation in protection by BCG: implications of and for heterologous immunity. Lancet, 1995,346:1339-1345.
[8] Skeiky Y A, Sadoff J C. Advances in tuberculosis vaccine strategies. Nat Rev Microbiol, 2006,4:469-476.
[9] Mahairas G G, Sabo P J, Hickey M J. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. Bacteriology, 1996,178:1274-1282.
[10] Skjot R L, Oettinger T, Rosenkrands I, Comparative evaluation of Low-molecularmass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T cell antigens. Infect Immun, 2000,68:214-220.
[11] Brusasca P N, Peters R L, Motzel S L. Gennaro, Antigen recognition by serum antibodies in non-human primates experimentally infected with Mycobacterium tuberculosis, Comp Med, 2003,53:165-172.
[12] Kamath A T, Feng C G, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacteria tuberculosis. Infect Immun, 1999, 67(4): 1702-1707.
[13] Smith G E, Fraser M J, Summers M D. Molecular engineering of the Autographa californica nuclear polyhedrosis virus genome: deletion mutations within the polyhedrin gene. Journal of Virology, 1983, 46(2): 584-593.
[14] Schmidt F R. Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol, 2004, 65: 363-372.
[15] Blanchard P, Mahe D, Cariolet R, et al. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine cirocovirus type 2 (PCV2) proteins. Vaccine, 2003, 21: 4565-4575.
[16] Cox M J, Hollister J R. FluBlok. a next generation influenza vaccine manufactured in insect cells. Biologicals, 2009, 37: 182-189.
[17] Centers for Disease Control and Prevention. Development of new vaccines for tuberculosis. Recommendations of the Advisory Council for the Elimination of Tuberculosis (ACET). Morb Mortal Wkly Rep, 1998, 47: 1-6.
[18] Floyd K, Blanc L, Raviglione M, et al. Resources required for global tuberculosis control. Science, 2002, 295(3): 2040-2041.
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