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[Preparation of rabbit anti-recombinant human calreticulin antibody and its characterization]
Xiao-lei Tang1, Xiao-dong Liang, Rui-huan Xu
1Department of Clinical Immunology, Guangdong Medical College, Dongguan 523808, China.
Aim:
To prepare the rabbit anti-recombinant human calreticulin (hCRT) antibody and its characterization.
Methods:
The gene coding for hCRT was amplified by PCR and cloned into prokaryotic expression vector pET32a. Then the recombinant plasmid pET32a/hCRT was transformed into E.coli BL21 (DE3) and expressed under IPTG induction. The recombinant hCRT was purified through Ni(2+) -NT agarose gel column and the purified hCRT used as immunogen to immunize the rabbit.The titer and specificity of the rabbit anti-hCRT antibody were analyzed by ELISA, Western blot and immunohistochemical staining, respectively.
Results:
The recombinant hCRT was successfully expressed and purified, and the polyclonal anit-hCRT antibody was successfully prepared. The titer of the antiserum was 1:51 200 by ELISA. Western blot analysis showed that the antibody reacted specifically to hCRT. Immunohistochemical staining detection manifested the antibody could recognize the native hCRT.
Conclusion:
The rabbit anti-hCRT antibody with high titer and specificity has been successfully prepared, which lays the foundation for further research on detection of hCRT and its clinical application.
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