高等学校化学学报

高等学校化学学报 ›› 2011, Vol. 32 ›› Issue (1): 95.

• 研究论文 • 上一篇    下一篇

新型抗HIV-1蛋白GRFT的构建、表达及活性研究

李昌1,李霄1,刘玉生1,于芳1,韩佳丽2,胡博1,叶明3,王婧1,杜寿文1,金宁一1   

  1. 1.  军事医学科学院解放军基因工程实验室, 长春 130062; 
    2.  沈阳农业大学畜牧兽医学院,  沈阳 110161; 
    3.  延边大学农学院,  延吉133400
  • 收稿日期:2010-03-29 修回日期:2010-06-28 出版日期:2011-01-10 发布日期:2010-12-11
  • 通讯作者: 金宁一 E-mail:ningyij@hotmail.com
  • 基金资助:

    国家重大传染病专项基金(批准号:   2008ZX10004-015)、军内十一五科技攻关项目(批准号: 06G127)、吉林省高新技术产业发展项目和长春市科技特派员行动计划项目资助.

Construction, Expression and Activities of a Novel Anti-HIV-1 Protein GRFT

LI Chang1, LI Xiao1, LIU Yu-Sheng1, YU Fang1, HAN Jia-Li2, HU Bo1, YE Ming3, WANG Jing1, DU Shou-Wen1, JIN Ning-Yi1*   

  1. 1. Laboratory of Genetic Engineering of PLA, Academy of Military Medical Sciences, Changchun 130062, China;
    2. College of Animal Science and Veterinary Medicine,  Shenyang Agricultral University, Shenyang 110161, China;
    3. Agricultural College of Yanbian University, Yanji 133400, China
  • Received:2010-03-29 Revised:2010-06-28 Online:2011-01-10 Published:2010-12-11
  • Contact: Jin Ning-yi E-mail:ningyij@hotmail.com
  • Supported by:

    国家重大传染病专项基金(批准号:   2008ZX10004-015)、军内十一五科技攻关项目(批准号: 06G127)、吉林省高新技术产业发展项目和长春市科技特派员行动计划项目资助.

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被引次数

14

摘要： 根据已知的新型抗HIV-1蛋白GRFT基因氨基酸序列，推测其DNA编码序列，密码子优化及修饰后进行全基因化学合成，连接到原核表达载体pET28a（+）中，转化大肠杆菌BL21(DE3)，IPTG诱导表达，获得目的蛋白. SDS-PAGE、Western Blot分析结果表明，目的蛋白得到良好表达并具有抗原活性；对表达条件进行优化，在最佳表达条件下，目的蛋白的表达量可占菌体总蛋白的55.84%；利用Ni2+-NTA柱亲和层析法进行目的蛋白的复性和纯化，凝胶成像系统扫描分析表明，纯度可达94.06%；运用Dot-ELISA法进行复性蛋白的抗原结合活性检测，结果显示，目的蛋白能够与HIV-1膜蛋白抗原特异性结合，初步证明所表达的重组蛋白具有良好的体外结合活性；基于制备的能够表达HIV-1 gp120基因的靶细胞模型，运用IFA法开展目的蛋白的细胞结合活性研究，结果显示，目的蛋白能够与靶细胞发生特异性反应，表明成功制备并获得了具有生物活性的新型抗HIV-1蛋白GRFT，为进一步研制新型抗HIV-1基因工程药物及其靶向治疗制剂奠定了坚实基础.

关键词: HIV-1, 抗病毒蛋白, GRFT, 表达, 生物活性

Abstract: A DNA sequence encoding GRFT was synthesized according to the deduced amino acid sequence of GRFT, using an E. coli codon preference table, and cloned into the prokaryotic expression vector pET28a (+). Then the recombinant plasmid was transfered into E.coli BL21(DE3). The target protein was obtained after induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The results of SDS-PAGE and Western Blot showed that the protein was well expressed and had antigenic activities. When different parameters were optimized, the highest production could account for 55.84% of total bacterial protein. Gel scan analysis showed that the purity was 94.06% after the target protein refolding and purification with Ni2+-NTA affinity chromatography. The antigenic and cellular biding activities of target protein were identified by Dot-ELISA and IFA methods based on the HIV-1 infection target cell models prepared by our laboratory. The results suggested that the recombinant protein has special recognition and binding activity with target cells. This research will settle solid foundation for further investigate of anti-HIV-1 genetic engineering agents and targeted therapy.

Key words: HIV-1, Anti virus protein, Griffithsin, Expression, Biological activities

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