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Construction and expression of hepatocellular carcinoma-associated antigen H2-calponin
FAN Sheng-long,HUANG Peng,TU Zhen-bo,LI Ling,ZHANG Sheng-chang,LU Qiao-qiao,ZHAO He,ZHOU Su-fang
2012, 4 (4):
311-315.
doi: 10.3969/j.issn.1674-5671.2012.04.03
Objective To construct a pCold III-hepatocellular carcinoma-associated antigen H2-calponin（CNN2） expression plasmid， and to express and purify CNN2 from bacteria. Methods CNN2 cDNA was amplified and constructed into the pCold III expression vector.The resulting pCold III-CNN2 expression plasmid was transformed into E. coli BL21（DE3） pLysS；optimal conditions for culturing E. coli BL21（DE3） pLysS/pCold III-CNN2 and for inducing CNN2 expression were explored.SDS-PAGE was used to track the HIS-CNN2 fusion， which was purified on Ni-NTA resin. The identity of the purified protein was checked by Western blotting with anti-CNN2 antibody. Results CNN2 protein was overexpressed in E.coli BL21（DE3） pLysS/pCold III-CNN2.Under optimal culture and induction conditions，the expressed protein HIS-CNN2 accounted for 35% of total bacterial protein. The fusion protein was found in both the sol-uble and insoluble fractions of cell lysate，indicating that CNN2 protein was expressed in soluble form.Western blotting with anti-CNN2 antibody detected the purified His-CNN2 fusion，suggesting that the overexpressed protein retains its immunogenicity. Conclusion The pCold III-CNN2 expression plasmid was successfully constructed and antigenic CNN2 protein was obtained.
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