The Study On The Function Of The Human Non-muscle Myosin Light Chain Kinase In The Course Of The Proliferation Of Cancer Cells

Objective To construct the recombinant vector which can express human non-muscle myosin light chain kinase (hnmMLCK) N-terminal in E.Coli, express and purify the target protein in vitro and prepare the polyclonal antibody. To study the function of MLCK on cell proliferation with the specific MLCK inhibitor, ML-9(l-(5-chloronaphthalene-l-sulfonyl)-lh-hexahydro-l,4-diazepine), and get the information of the MLCK expression of the cells by immune fluorescence technique. Methods PCR was used to amplify hnmMLCK N-terminal cDNA, and the product was ligated into the T-Vector to get the recombinant plasmid pGEM-hnmMLCK. After proved by restrictive endonuclease mapping, the recombinant plasmid was amplified, digested by the restriction endonuclease( EcoRI and Hind III ). And the target gene, hnmMLCK, was inserted into pBKcmv to construct expression vector. Positive clones were screened by blue-white color screen and antibiotic resistance, and were proved by restrictive endonuclease mapping analysis and DNA sequencing. IPTG was used to induce the expression. Gather the special bands from the gels after SDS-PAGE. Immune the New Zealand rabbit with the target protein which was purified by elctroelution to generate the antibody and measure the tilter of the polyclonal antibody by immnodiffusion. To treat the human bladder cancer cell line ScaBer with different concentrations of MLCK specific inhibitor, ML-9. P-ni trophnyl -phosphate was used to test the cell proliferation. We checked the expression of MLCK of the ScaBer cells pretreated by different concentrations of ML-9 by immune fluorescence technique with rabbit anti-hnmMLCK antiserum, the primary antibody, and donkey anti-rabbit antibody( Fluorescein isothiocyanate, FITC), the secondary antibody. To prepare genomic DNA of the cells to check whether the apoptosis by electrophoresis. Results Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the constructed plasmid was the recombinant one. SDS-PAGE showed the expressedproduct was about 21kD and the quantity was about 21% of the whole protein of the bacteria. The tilter of the polyclonal antibody is 1:16. The proliferation was inhibited significantly (P<0.01) when the concentration of ML-9 got to 10 MM. There are morphological changes between the control cells and the cells pretreated. The expression of MLCK was increased when the concentration of ML-9 , the specific MLCK inhibitor , was up to 30 M M by immune fluorescence technique. ML-9 induced neither apoptosis nor necrosis in the bladder cancer cells. Conclusion The expressed plasmid was constructed, hnmMLCK was expressed in E.Coli, and the polyclonal antibody was prepared successfully. Using the polyclonal antibody, we can check the expression of the MLCK by immune fluorescence technique. ML-9 can decreased the proliferation of the human bladder cancer cell line ScaBer without apoptosis.