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人黑素瘤相关抗原D4原核表达条件的优化 预览

Optimization of prokaryotic expression of human melanoma-associated antigen D4
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摘要 目的:探索黑素瘤相关抗原D4(MAGE-D4)基因工程菌原核诱导表达的最佳条件,以提高目的蛋白产量。方法:设置不同宿主菌(DH5α、TB1、Rosetta)、诱导时机(重组菌分别培养0.5~3h后进行诱导)、诱导剂IPTG浓度(0.1~0.9mmol/L)和诱导时间(1~8h)等条件,诱导MAGE-D4重组质粒表达,通过SDS-PAGE电泳和ImageJ软件扫描电泳图谱的方法,对目的蛋白的表达量和可溶性进行分析。通过间接酶联免疫吸附试验(ELISA)法对35例肝癌患者血清MAGE-D4抗体进行检测,分析MAGE-D4融合蛋白的免疫活性。结果:将重组质粒转化至Rosetta(DE3)大肠杆菌,菌液OD600达到0.8时加入IPTG至终浓度0.9mmol/L,37℃诱导培养7h,可有效提高目的蛋白的表达量。可溶性MAGE-D4融合蛋白表达量约占菌体总蛋白的25.5%。肝癌患者血清中MAGE-D4抗体的阳性率为28.57%(10/35),其MAGE-D4抗体平均效价明显高于正常人(P<0.05)。结论:本研究优化了MAGE-D4融合蛋白的原核表达条件,获得了可溶性、具有免疫活性的重组蛋白。 Objective: To explore the optimal conditions for prokaryotic expression of melanoma-associated antigen D4 (MAGE-D4) gene engineering bacteria,and to enhance the production of target protein. Methods: MAGE-D4 recombinant plasmid was induced by different host bacteria (DH5α,TB1,Rosetta),starting time of induction (recombinant bacteria were induced after cultured for 0.5-3 hours),IPTG concentration (0.1-0.9 mmol/L) and induction time (1-8 hours).The expression and solubility of the target protein were analyzed by SDS-PAGE electrophoresis and Image J software scanning.MAGE-D4 antibody in serum of 35 patients with hepatocellular carcinoma was detected by indirect ELISA to identify the immune activity of MAGE-D4 fusion protein. Results: The recombinant plasmid was transformed into Escherichia coli Rosetta (DE3).0.9 mmol/L IPTG was added when the OD 600 reached 0.8 and induced at 37 ℃ for 7 hours,which could effectively increase the expression of the target protein.The soluble MAGE-D4 fusion protein accounted for about 25.5% of the total cellular proteins.The positive rate of MAGE-D4 antibody in serum of hepatocellular carcinoma was 28.57%(10/35),The average titer of MAGE-D4 antibody in serum of patients was significantly higher than that in normal( P <0.05). Conclusion: In this study,the prokaryotic expression conditions of MAGE-D4 fusion protein were optimized,and the soluble recombinant protein with immunologic activity was obtained.
作者 邓芸婷 顾永耀 蔡丹昭 罗育 高精洧 李霞琼 黄冠文 谢小薰 贺菽嘉 Deng Yunting;Gu Yongyao;Cai Danzhao;Luo Yu;Gao Jingwei;Li Xiaqiong;Huang Guanwen;Xie Xiaoxun;He Shujia(Department of Biochemistry and Molecular Biology,Guangxi Medical University,Nanning 530021,China;Department of Pathology,Guangxi Medical University,Nanning 530021,China;Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research,Guangxi Medical University,Nanning 530021,China;Guangxi Medical University,Nanning 530021,China;Department of Histology and Embryology,Guangxi Medical University,Nanning 530021,China)
出处 《广西医科大学学报》 CAS 2019年第7期1050-1055,共6页 Journal of Guangxi Medical University
基金 国家自然科学地区基金资助项目(No.81660464) 广西高校科学技术研究课题资助项目(No.KY2015YB058) 广西医科大学青年科学基金资助项目(No.GXMUYSF2014012) 广西高校生物分子医学研究重点实验室开放课题资助项目(No.GXBMR201601).
关键词 黑素瘤相关抗原D4 原核表达 优化 melanoma-associated antigen D4 (MAGE-D4) prokaryotic expression optimization
作者简介 通信作者:贺菽嘉,E-mail:heshujia@gxmu.edu.cn.
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