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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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