Expression of mice interlecukin-23 in pichia and preliminary studies on its biology function / 中国免疫学杂志
Chinese Journal of Immunology
;
(12): 120-123, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-403917
ABSTRACT
Objective:
To clone the mice interleukin-23(mIL-23) and express it in Pichia efficiently.Methods:
Two subunits of IL-23 were amplified by PCR from pcDNA3/mIL-23,and ligated together with adaptor by over-PCR.The IL-23 cDNA confirmed by sequencing was inserted into expressing vector pPICZαA and expressed in Pichia X-33 strain.IL-23 protein expression was induced by methanol and ammonia.The recombinant IL-23 was identified by immunoblot and its biological activity was analyzed.Results:
DNA sequencing confirmed that cloned cDNA was identical to the published sequence of mIL-23.The recombinant plasmid pPICZαA/mIL-23 was electroprated into X-33.An expected 72 kD protein of mIL-23 was founded both in the induced pellets and supermatants by SDS-PAGE and coomassie blue staining.The 72 kD protein could be recognized in Western blot.While we could detect bands at 72 kD in cell pellets induced more than 24 h by Western blot.Detected by Western blot using anti-His antibody,we could see positive band at 72 kD from supernatants induced 24 and 36 h.While we could detect band at 72 kD in cell pellets induced between 24 h and 96 h.Meanwhile,we could see obviously proliferation of mice peripheral blood mononuclear cells(PBMC) treated with the induced pellets and supermanants.Conclusion:
We have successfully expressed mIL-23 protein in Pichia and the expressed product has its bioactivity in promoting the proliferation on PBMC.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Immunology
Year:
2010
Type:
Article
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