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[Preparation of monoclonal antibody against human hNudC and its expression in human megakaryocytes].

AIM
To prepare and characterize the monoclonal antibody (mAB) against human nuclear distribution C (hNudC).


METHODS
The coding region of hNudC was amplified from human embryo liver tissue and the recombinant prokaryotic expression vector pET28b was constructed. hNudC protein was expressed as a fusion protein with an N-terminal 6-His tag. The purified recombinant hNudC protein was used to immunize BALB/c mice for preparing mAb. The hybridoma cells produced from mAB were screened by ELISA and the specificity of mAb was analyzed by immunohistochemical staining and Western blot.


RESULTS
Two hybridoma cells (2C16 and 2D8) secreting mAb against hNudC were developed. The isotypes of the two mABs were IgGI and IgM, respectively. ELISA detection showed that the titer of mABs, 2C16 and 2D8 was 1:64, 1:32 in cultured supernatant and 1:1 x 10(5), 1:5 x 10(4) in ascites, respectively. They could specifically bind to recombinant hNudC. The results of immunohistochemical staining and Western blot indicated that mAb could specifically recognize hNudC in human Dami, Meg-01 cell lines and human marrow CD41+ megakaryocytes generated from umbilical core blood.


CONCLUSION
Monoclonal antibodies against hNudC with high titers and specificity have been successfully prepared, which will be useful for assessing the function, native distribution and aberrant expression of hNudC protein.