Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica

Acta Academiae Medicinae Sinica ›› 2009, Vol. 31 ›› Issue (4): 403-409.doi: 10.3881/j.issn.1000-503X.2009.04.004

• Original Articles • Previous Articles     Next Articles

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

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