title: [Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice].
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 beta-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-gamma); 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-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/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-gamma 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.
authors.affiliation: Department of Virology, School of Public Health, Shandong University, Jinan 250012, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Miao
authors.initials: M
authors.lastname: Xu
authors.forename: Wei-Xin
authors.initials: WX
authors.lastname: Du
authors.forename: Bao-Wen
authors.initials: BW
authors.lastname: Chen
authors.forename: Zhi-Yu
authors.initials: ZY
authors.lastname: Wang
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Wang
authors.forename: Na
authors.initials: N
authors.lastname: Dong
authors.forename: Cheng
authors.initials: C
authors.lastname: Su
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Shen
authors.forename: Guo-Zhi
authors.initials: GZ
authors.lastname: Wang
mesh_terms: D000217:Acyltransferases / Q000008:administration & dosage / Q000302:isolation & purification* / Q000627:therapeutic use; D000276:Adjuvants, Immunologic / Q000627:therapeutic use*; D000818:Animals; D000942:Antigens, Bacterial / Q000008:administration & dosage / Q000302:isolation & purification* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000302:isolation & purification* / Q000627:therapeutic use; D004926:Escherichia coli; D007111:Immunity, Cellular; D007371:Interferon-gamma; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000627:therapeutic use
chemical_list: D000276:Adjuvants, Immunologic; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C118127:HspX protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins; D007371:Interferon-gamma; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis