[Prokaryotic expression of human mast cell chymase gene and preparation of its polyclonal antibody].
OBJECTIVE
To express the human mast cell chymase cDNA in E.coli and prepare the antibody against human mast cell chymase with recombinant chymase.


METHODS
The human mast cell chymase cDNA was cloned by RT-PCR. The recombinant chymase was expressed in E.coli with L-Arabinose induction and purified by Ni-NTA agarose column. Then the purified chymase was used as immunogen to immunize the rabbit. The titer and specificity of the anti-chymase antibody from the rabbit were analyzed by indirect ELISA and Western blot, respectively.


RESULTS
The recombinant chymase was successfully expressed in E.coli, and the polyclonal anit-chymase antibody was prepared by immunizing the rabbit with the purified recombinant chymase. The titer of the generated antiserum was detected to be 1:12 800 by ELISA. Western blot analysis showed this antibody bound specifically with chymase.


CONCLUSIONS
The anti-chymase antibody from the rabbit with high titer and specificity has been prepared with purified recombinant chymase as immunogen, which lays a foundation for further research into detection and function of chymase.
Chen
Zhang-Quan
ZQ
Department of Clinical Immunology, Guangdong Medical College, Zhanjiang 524023, China.
Huang
Zhen
Z
He
Tian-Wen
TW
Lu
Tian-Tian
TT
Liang
Xiao-Dong
XD
He
Shao-Heng
SH
D000918:Antibody Specificity; D015153:Blotting, Western; D053818:Chymases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007106:Immune Sera / Q000032:analysis / Q000276:immunology*; D008407:Mast Cells / Q000201:enzymology*
D018076:DNA, Complementary; D007106:Immune Sera; D053818:Chymases