CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定

doi:10.13523/j.cb.20140604

中国生物工程杂志

2014, Vol. 34

Issue (06): 23-30 DOI: 10.13523/j.cb.20140604

研究报告

CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定

徐丹^1,2,4, 刘敏^1,2, 孔菊³, 李校堃^1,2, 姜潮^1,2

1. 温州医科大学浙江省生物技术与制药工程重点实验室温州 325035;
2. 吉林农业大学生物反应器与药物开发教育部工程研究中心长春 130118;
3. 巨野县人民医院菏泽 274900;
4. 济南康和医药科技有限公司济南 250101

Expression, Purification and Immunogenicity Analysis of Recombinant CFP10-ESAT-6-MPB64 Using the Baculovirus Expression System

XU Dan^1,2,4, LIU Min^1,2, KONG Ju³, LI Xiao-kun^1,2, JIANG Chao^1,2

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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摘要：

目的：在杆状病毒昆虫细胞表达系统（baculovirus expression vector system，BEVS）中表达结核分枝杆菌蛋白CFP10-ESAT-6-MPB64，并鉴定其免疫原性。方法：目的基因CFP10-ESAT-6-MPB64连接到pFastBac转移载体并转化DH10Bac感受态，通过Tn7转座片段将目的基因转座到Bacmid中，得到Bacmid-CFP10-ESAT-6-MPB64穿梭载体，脂质体包被后转染Spodoptera frugiperda（Sf9）细胞收获病毒，病毒转染细胞后收集上清通过Co亲和层析纯化得到目的蛋白。纯化的蛋白免疫Balb/c小鼠并检测血清中特异性抗体滴度及PPD抗体，ELISA检测CFP10-ESAT-6-MPB64蛋白刺激脾脏细胞产生IFN-γ的浓度，MTT法检测目的蛋白对免疫后小鼠脾脏细胞的增殖作用。结果：CFP10-ESAT-6-MPB64在昆虫细胞中成功表达，纯化后蛋白纯度达90%以上，蛋白产量为42mg/L，纯化蛋白能有效刺激Balb/c小鼠产生抗体，提高小鼠脾脏细胞培养基中IFN-γ的含量，目的蛋白在1~50μg/ml之间对脾脏细胞有明显的促增殖作用。

关键词： BEVS; Sf9细胞; 结核分枝杆菌

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.

Key words: BEVS Sf9 Mycobacterium tuberculosis

收稿日期: 2014-05-06 出版日期: 2014-06-25

ZTFLH:

R392.11

基金资助:

国家“863”计划（SQ2011AA100606）、浙江省自然科学基金（LY13H300006）、2012年省级重点科技创新团队项目（2012R10042-10）、2012年吉林省高层次创新创业引进人才项目资助项目

通讯作者: 李校堃, 姜潮 E-mail: chaojiang10@hotmail.com

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引用本文:

徐丹, 刘敏, 孔菊, 李校堃, 姜潮. CFP10-ESAT-6-MPB64在杆状病毒系统中的表达纯化及免疫原性鉴定[J]. 中国生物工程杂志, 2014, 34(06): 23-30.

XU Dan, LIU Min, KONG Ju, LI Xiao-kun, JIANG Chao. Expression, Purification and Immunogenicity Analysis of Recombinant CFP10-ESAT-6-MPB64 Using the Baculovirus Expression System. China Biotechnology, 2014, 34(06): 23-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140604 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I06/23

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