[Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice].
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.
Chen
Lei
L
Department of Virology, School of Public Health, Shandong University, Jinan 250012, China.
Xu
Miao
M
Du
Wei-Xin
WX
Chen
Bao-Wen
BW
Wang
Zhi-Yu
ZY
Wang
Ya-Jun
YJ
Dong
Na
N
Su
Cheng
C
Shen
Xiao-Bing
XB
Wang
Guo-Zhi
GZ
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
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