高等学校化学学报

高等学校化学学报

• 研究论文 • 上一篇    下一篇

新型抗HIV-1重组导向制剂SL41在毕赤酵母中的表达及活性实验

李昌1,2, 王宏3, 金宁一1, 金洪涛1,2, 刘玉生1,2, 于芳1, 李子健1, 张立树1   

    1. 军事医学科学院军事兽医研究所, 全军基因工程重点实验室, 长春 130062;
    2. 吉林大学农学部畜牧兽医学院, 长春 130062;
    3. 暨南大学生命科学技术学院, 广州 510632
  • 收稿日期:2006-11-06 修回日期:1900-01-01 出版日期:2007-08-10 发布日期:2007-08-10
  • 通讯作者: 金宁一

Expression and Activities of Recombinant Directing Agent SL41 Against HIV-1 in Pichia pastoris

LI Chang1,2, WANG Hong3, JIN Ning-Yi1*, JIN Hong-Tao1,2, LIU Yu-Sheng1,2, YU Fang1, LI Zi-Jian1, ZHANG Li-Shu1   

    1. Laboratory of Genetic Engineering of PLA, Academy of Military Medical Sciences, Changchun 130062, China;
    2. College of Animal Sciences and Veterinary Medicine, Department of Agronony, Jilin University, Changchun 130062, China;
    3. College of Life Science and Technology, Jinan University, Guangzhou 510632, China
  • Received:2006-11-06 Revised:1900-01-01 Online:2007-08-10 Published:2007-08-10
  • Contact: JIN Ning-Yi

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被引次数

21

摘要： 以酵母分泌型表达载体pPIC9k为基础, 通过一段柔性连接肽Linker构建含有人源化抗HIV-1 gp41单链抗体(ScFv41)和免疫诱导因子葡萄球菌肠毒素A(staphylococcal enterotoxin A, SEA)的融合表达质粒pPIC9k-SL41, 线性化后, 采用电转化法整合入巴斯德菌毕赤酵母GS115中, 经His+MutS表型鉴定、PCR分析以及G418筛选获得高拷贝重组转化子. 摇瓶培养、甲醇诱导表达、SDS-PAGE和Western Blot分析结果表明, 目的蛋白得到良好表达, 表达量最高可达到47.9 mg/L. 目的蛋白经初步纯化后, 用于制备的HIV-1感染靶细胞复制模型进行抗体亲和力测定、细胞结合活性测定和细胞杀伤活性研究, 结果显示, 目的蛋白能够很好地与靶细胞模型中的HIV-1外膜蛋白gp160发生结合反应, 并可介导特异的CTL反应, 对靶细胞具有明显的杀伤活性, 表明获得了具有生物活性的抗HIV-1重组导向制剂.

关键词: HIV-1, 重组导向制剂, 单链抗体, 金黄色葡萄球菌肠毒素A(SEA), 分泌表达, 毕赤酵母

Abstract: The humanize anti-HIV-1 single chain Fv fragment(ScFv) gp41 gene and super antigen staphylococcal exterotoxin A(SEA) gene were fused via soft linker peptide, and cloned into expression vector pPIC9k. The recombinant plasmid was linearized and transferred into Pichia pastoris strains GS115 by electroporation. High copies of trransformants were obtained with Muts and Mut+ phenotype identification, PCR amplification and screening of G418. After flask culture and inducing expression by methanol, the targeted protein was well expressed and performed via SDS-PAGE and Western Blot. The highest production was 47.9 mg/L when different parameters were optimized. The primarily-purified protein was analyzed with antibody affinity assay, cellular binding activity and cellular killing activity using the HIV-1 infection target cell models prepared by our laboratory. The results suggest that the constructed anti-HIV-1 gp41 recombinant agent SL41 has a good biological activity and specific CTL cytotoxicity activity. This research will provide a potential values for AIDS treatment and the settled solid foundation for clinical trials.

Key words: HIV-1, Recombinant directing agent, Single chain Fv fragment, Staphylocoecus extoxin A(SEA), Secrete expression, Pichia pastoris

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