Expression Of OPCML Gene In Insect Cells And Studies Of Its Activity

It is proved that the OPCML gene is locate at 11q25,LOH (loss-of-heterozygosity)frequently can be found in this region.OPCML contains seven extrons and spans 600 kilobasees,molecular weight is about 60kd,which protein expression in cell membranes of opioid receptors and belong to cell adhension factor.OPCML protein exist widely in nervous system cells and plays an important role in the regulation on opioid receptors which promote cell differentiation and changed the membrane properties. In decades,the vast majority of research mainly concentrated in the central nervous system,recognition and signaling between the cells adhesion,and play a role through activating adenosine acid ring of enzyme (AC) and related ion channels.The researches had confirm human chromosomes of loss 11q25 LOH (with heterozygosity) in the epithelial ovarian cancer was positively related to the adverse outcomes.Therefore,Thus speculate in this region existence the epithelial ovarian cancer related tumor-suppressor genes (TSG),OPCML is one of the candidates TSG. This study uses baculovirus infect SF9 insect cell,appraisal the expression of OPCML gene in SF9 insect cell,and its expression conditions are preliminary exploration. Materials and methodsExperiment 1:opcml gene amplification and the construction of insect expression vector carry the opcml gene,Find out the opcml gene sequences in GenBank and design primer, using the polymerase chain reaction (RT-PCR) to amplificate opcml genes and connected to baculovirus carrier PACGp67A plasmid,get OPCML—PACGp67A plasmid, as preparing the next chapter opcml genes transfection the insect cell.Experiment 2:Expression of OPCML Gene in Insect CellsChoose good growth state SF9 insect cell, infect the SF9 insect cell with the restructuring OPCML—PACGp67A plasmidsindbis, observation infection symptom; Using PCR, enzyme cutting technology testing OPCML gene transcription; SDS-page analysis utrient-containing medium after infection if there is specific protein strip and the molecular size ; With different doses of virus, infection SF9 cell liquid infection, choose the duration of quantity of different high infection time expressed as best expression time; Using the Western blot to inspection opcml gene in insect cells.Experiment 3:The vitro studies of OPCML in ovarian cancer cell proliferation and adhesion,growth cycle in vitro studiesDetection of the function index,the growth curve,the proliferation of the cell cycle and other biological behavior changes which the purification of OPCML protein expression in great quantities from Insect cell ,in vitro direct role in the epithelial ovarian cancer cells A2780. ResultsResearch results show that opcml gene expression in SF9 insect cells after combinant baculovirus infect the SF9 insect cel.It is prove that the opcml protein succeed expression in baculovirus system and the infection time should be controlled in 5-6 days.Streaming cell count determine the cell cycle results showed that OPCML protein does not affect the growth period of A2780 cells,but cellular adhesive experimental results show that OPCML protein can improve the A2780 cell adhesion strength,but must have enough protein concentration.Conclusion1.The cloned OPCML gene open reading sequence, and successfully build eukaryotic expression plasmid OPCML—PACGp67A reorganization.2.The SF9 insects cells which infected by the reorganization virus could efficiently express OPCML proteins,after purification ,verifying the protein by Western blot .3.OPCML protein on A2780 cell proliferation without inhibition, but can increase the adhesion between cells.4.OPCML as a tumor-suppressor gene targeting treatment of ovarian, there may exist great potential.