中国医学科学院学报

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中国医学科学院学报

中国医学科学院学报 ›› 2009, Vol. 31 ›› Issue (4): 403-409.doi: 10.3881/j.issn.1000-503X.2009.04.004

• 论著 • 上一篇    下一篇

结核分枝杆菌H37Rv两种抗原Ag85b、HspX的制备及其与佐剂的联合应用

陈磊1;徐苗2;都伟欣2;陈保文2;王志玉1;王雅君2;董娜3;苏城2;沈小兵2;王国治2   

  1. 1山东大学公共卫生学院病毒学研究室, 济南 250012
    2中国药品生物制品检定所细菌一室, 北京 100050
    3中国医学科学院北京协和医学院实验动物研究所, 北京 100021
  • 收稿日期:2009-04-01 修回日期:1900-01-01 出版日期:2009-08-30 发布日期:2009-08-30
  • 通讯作者: 王国治

Preparation of Two Antigens - Ag85b and HspX of Mycobacterium Tuberculosis H37Rv and the Effects of Their Co-administration with Adjuvants in Mice

CHEN Lei1; XU Miao2; DU Wei-xin2;CHEN Bao-wen2; WANG Zhi-yu1; WANG Ya-jun2; DONG Na3; SU Cheng2; SHEN Xiao-bing2; WANG Guo-zhi2   

  1. 1Department of Virology,School of Public Health, Shandong University, Jinan 250012,China
    21st Bacterial Vaccine Division, National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China
    3Institute of Laboratory Animal Science, CAMS and PUMC, Beijing 100021,China
  • Received:2009-04-01 Revised:1900-01-01 Online:2009-08-30 Published:2009-08-30
  • Contact: WANG Guo-zhi

摘要: 摘要:目的 用分子生物学方法制备H37Rv的两种抗原Ag85b、HspX,通过与铝佐剂和CpG佐剂联用免疫小鼠进行初步药理学实验以观察其生物学活性。方法 常规方法分别构建重组表达质粒pET30a-Ag85b和pET30a-HspX,内切酶鉴定目的片段后两种质粒分别转化BL-21大肠杆菌,经异丙基-β-D-硫代半乳糖苷诱导后分别产生两种蛋白;用阴离子交换柱Source30、QHP以及疏水层析柱对两种蛋白进行纯化;蛋白测序进行鉴定。纯化后的两种蛋白分别与铝佐剂和CpG佐剂联用(Ag85b,Ag85b+Al,Ag85b+CpG,Ag85b+Al+CpG;HspX,HspX+Al,HspX+CpG, HspX +Al+CpG),免疫C57/BL小鼠,同时设置生理盐水对照组,取脾细胞进行酶联免疫斑点实验(ELISPOT)检测干扰素-γ(IFN-γ)的分泌;同时进行淋巴细胞增殖实验检测脾淋巴细胞经体外刺激后的增殖情况。结果 成功构建了两种重组表达质粒pET30a-Ag85b和pET30a-HspX,成功诱导表达了两种蛋白;经过纯化,两种蛋白纯度均达到95%;经鉴定两种蛋白纯化产物的N端15个氨基酸序列与目的序列相同;Ag85b+CpG、Ag85b+Al和Ag85b+CpG+Al组经Ag85b(80μg/ml)体外刺激后分泌IFN-γ显著升高,与生理盐水组比较差异具有显著性(P<0.05);HspX+CpG+Al组经HspX(80μg/ml)体外刺激后分泌IFN-γ显著升高,与生理盐水组比较差异具有显著性(P<0.05)。结论 成功表达并纯化了结核分枝杆菌H37Rv的两种抗原Ag85b和HspX,与铝佐剂和CpG佐剂联用后成功刺激小鼠产生了细胞免疫反应,证实了两种重组蛋白的生物学活性,为下一步药效学评估奠定了基础。

关键词: 结核分枝杆菌, 重组蛋白质, 佐剂, Ag85b, HspX

Abstract: ABSTRACT:Objective To synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice. Methods Recombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl β-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b,Ag85b+Al,Ag85b+CpG,Ag85b+Al+CpG;HspX,HspX+Al,HspX+CpG, HspX+Al+CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-γ); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens. Results The purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-γ from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80μg/ml) remarkably increased in Ag85b+CpG group, Ag85b+Al group, and Ag85b+CpG+Al group; the changes were significantly different between these three groups and control group(P<0.05). For HspX, the changes were significantly different between HspX +Al+CpG group and normal sodium group, although remarked increase of IFN-γ was also observed in HspX group,HspX+Al group, and HspX+CpG group. Conclusions Ag85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.

Key words: mycobacterium tuberculosis, recombinant protein, adjuvant, ac85b, dspX