高等学校化学学报

高等学校化学学报 ›› 2011, Vol. 32 ›› Issue (1): 84.

• 研究论文 • 上一篇    下一篇

重组鼠疫汉坦钩端多抗原位点同源合成基因的表达及活性鉴定

浦昀1,2,齐燕飞1,徐国良3,徐卉3,张士尧1,孟日增1,2,宋秀玲1,杨怀宁2,李娟1   

  1. 1. 吉林大学公共卫生学院,  长春 130021;
    2. 吉林省出入境检验检疫局,  长春 130062;
    3. 吉林大学第一医院心血管中心,   长春 130021
  • 收稿日期:2010-06-13 修回日期:2010-10-08 出版日期:2011-01-10 发布日期:2010-12-11
  • 通讯作者: 李娟 E-mail:Li_juan@jlu.edu.cn
  • 基金资助:

    国家“重大新药创制”科技重大专项项目(批准号: 2009ZX09103-105)、高等学校博士学科点专项科研基金(批准号: 20090061120093)、国家质量监督检验检疫总局科技计划项目(批准号:  2009IK214)和吉林大学研究生创新研究计划项目(批准号:  20091028)资助.

Expression and Preliminary Activity Identification of Several Linked Antigen Epitopes of Consensce Genes of Yersinia Pestis, Hantavirus and Leptospira

PU Yun1,2, QI Yan-Fei1, XU Guo-Liang3, XU Hui3, ZHANG Shi-Yao1, MENG Ri-Zeng1,2, SONG Xiu-Ling1, YANG Huai-Ning2, LI Juan1*   

  1. 1. School of Public Health, Jilin University, Changchun 130021, China;
    2. Jilin Entry & Exit Inspection and Quarantine Bureau, Changchun  130062,   China;
    3.  Cardiovascular Center, First Hospital of Jilin University, Changchun 130021, China
  • Received:2010-06-13 Revised:2010-10-08 Online:2011-01-10 Published:2010-12-11
  • Contact: LI Juan E-mail:Li_juan@jlu.edu.cn
  • Supported by:

    国家“重大新药创制”科技重大专项项目(批准号: 2009ZX09103-105)、高等学校博士学科点专项科研基金(批准号: 20090061120093)、国家质量监督检验检疫总局科技计划项目(批准号:  2009IK214)和吉林大学研究生创新研究计划项目(批准号:  20091028)资助.

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被引次数

16

摘要： 利用人工利用人工合成含鼠疫耶尔森氏菌F1 capsule antigen的2个抗原位点，汉坦病毒的6个抗原位点和钩端螺旋体的6个抗原位点的串联基因片段，克隆到原核表达载体pET-20b中，构建重组表达质粒pET-rYHL.转化E.coliBL21(DE3)后获得表达菌株，表达菌株经IPTG诱导后，高效表达带组氨酸标签的以包涵体形式存在的融合蛋白。包涵体蛋白经尿素变性溶解、His Trap HP Kit柱纯化、SDS-PAGE及Western-blot分析表明, 在分子量40000处有一特异性蛋白条带，获得纯度达98%以上的蛋白。进一步ELISA 试验显示与鼠疫、流行性出血热及钩端螺旋体病阳性血清都能较好地结合, 而与其他鼠传疾病血清无交叉反应。

关键词: 鼠疫, 汉坦病毒, 钩端螺旋体, 合成基因, 基因表达, 活性鉴定

Abstract: The expression vector pET-rYHL was constructed by inserting the linked gene contained two antigen epitopes of F1 capsule antigen from Yersinia Pestis, six antigen epitopes of Hantavirus and six antigen epitopes of Leptospira into pET-20b and was identified by digestion with restriction enzymes and sequence analysis. Then an expression strain was selected after transformation of the recombined plasmid into E. coli BL21 (DE3), fusion protein with His-tag was efficiently expressed in the form of inclusion body after IPTG induction. The inclusion body was washed, dissolved and purified by Ni2+ chelate chromatography under denatured condition. SDS-PAGE analysis and Western blotting showed that the fusion protein with a molecular weight of about 40 000 was purified and the purity was up to 98%. The results ELISA showed specific reactions with Plague, Epidemic hemorrhagic fever and Leptospirosis positive sera respectively, and no cross-reaction with other positive sera sample(salmonella ) using the expression protein.

Key words: Plague, Hantavirus, Leptospira, Chimeric gene, Gene expression, Activity identification

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