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《Acta Biologiae Experimentalis Sinica》 2001-02
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LI Xiao Jun WU Jian Guo SI Jun Lin GUO Da Wen XU Jian Ping (Center of Medical Laboratory Science of PLA , Nanjing General Hospital of Nanjing Command , Nanjing ,210002)  
The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was used as template. After inserting the hCaM Ⅲ cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA - re-combinant expression vector (hCaM3/pBV220 ). The re-combinant plasmid was then transformed into E. coli DH5α. After heat induction, a high level expression of CaM protein was obtained. SDS - PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti - CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl - sepharose CL - 4B affinity chromatography from recombinant bacterial lysate. 3 - 4 mg of the purified protein were obtained from 1 liter of bacterial culture. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma). K562 cells and SP2 /O cells were seeded in 24 - well or 96 - well plate and cultured for 48h with rhCaM and CaM - antagonist trifluoperazine(TFP). Cell proliferation rates was determined by MTT assay. There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates. CaM- antagonist TFP had an inhibitory effect on cell proliferation rate. The inhibition could be corrected by the addition of extracellular rhCaM.
【Fund】: 江苏省自然科学基金
【CateGory Index】: Q26
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