Construction Of Eukaryotic Expression Vector、Establishment Of Lung Cancer Cell Strainand Function Of RASGRF2

RASGRF2is a rich multi-domain protein, compiled Code of1237amino acids, located on human5q13, allelic loss of the area of lung canceroften is considered a potential tumor suppressor gene. Our preliminary studyfound that RASGRF2loss of expression in lung cancer cells with abnormalmethylation of the relevant RASGRF2,obvious abnormal methylation inlung cancer. the purpose of this study is to construct RASGRF2-GFP fusiongene eukaryotic expression vector, observe the cellular localization ofproteins in lung cancer cells, establish stable expression RASGRF2lungcancer cell strain, and observed influnence on the cell cycle, which lay thefoundation of further study RASGRF2gene function.Part one RASGRF2-GFP fusion gene eukaryotic expression vectorand identification. Objective: to construct RASGRF2eukaryotic expressionvector tagged with GFP.Methods:pCMV6-GFP and RASGRF2-pCMV6-Myc-DDK were digested by SgfI and NotI restriction enzyme, thenRASGRF2was subcloned into the pCMV6-GFP vector to constructRASGRF2-pCMV6-GFP recombinant vector. Results: Enzyme digestion and sequencing data indicated that the recombinant vector was constructedexactly.Conclusion: The RASGRF2eukaryotic expression vector taggedwith GFP constructed successfully，which laid the foundation for furtherstudy RASGRF2gene function.Part two establishment of lung cancer cell strain of stable expressionRASGRF2and cellular localization and function. Objective: to establishstable expression RASGRF2lung cancer cell strain, and observedinflunence on the cell cycle and cellular localization.Methods:lung cancerH1299was transfected with RASGRF2-pCMV6-GFP and observed forlocation of RASGRF2protein under Inverted fluorescence microscope.Thecell strain of stable expressing RASGRF2was established by G418screencing,for flow cytometry cell cycle changes,and was determinedbyRT-PCR and Western blot. Results: The RASGRF2-pCMV6-GFPrecombinant protein was expressed successfully,and located cytoplasm inH1299cells by fluorescence microscope detection.The stable cell strainexpressed stably RASGRF2in H1299cells by RT-PCR, Western blotconfirmed. RASGRF can arrest cell cycle in S phase. Conclusion:estbalishment of the stable expression RASGRF2gene lung cancer cellsstrain, its protein cellular localization, has some influence on the cell cycleand found RASGRF2, which laid the foundation for further studyRASGRF2gene function in lung cancer.