Construction Of Human Synuclein-γ Eukaryotic Expression Vector And Its Stable Expression In Glioma Cells

Objective:There is many genes involved in the glioma's development process. They are interacting through a complex network, genes are the most basic unit of the network. Researching the Glioma on gene level,to further understanding the pathogenesis of glioma and the disease process has a fundamental help.Study a gene's variated regulation in gliomas has great significance in assessment for the diagnosis, prognosis and therapeutic target of glioma. To establish synuclein-γeukaryotic expression vector,and then use synuclein-y eukaryotic expression vector to construct stably overexpressed SNCG model in human glial tumor U373 cells, and to study how overexpression of SNCG influence on the U373 cell lines from gene,cell cycle and drug intervention three aspects.Methods:First, refer to GenBank database (NM₀03087), using Primer 5.0 software design primer SNCG,which is used to be amplified.Subsequently,with a template of plasmid pcDNA3-SNCG,SNCG full length gene was amplified by RT-PCR, of the amplified DNA fragments were purified, the product was purified eukaryotic expression vector pIRES2-EGFP were digested by the target band after electrophoresis, after recovery, with T4 DNA ligase and vector fragment were connected DH5αE. coli with the transformation of the bacteria into uniform coating containing kanamycin 50mg/L of the LB agar plate,37℃inverted culture 12 h, resistant colonies to be grown single colony picked after transfer to liquid medium in shake culture. By plasmid extraction kit instructions mention the small plasmid, recombinant plasmid as a template for PCR amplification of plasmid identification. BamH I and EcoR I and then the double-enzyme digestion cloned into eukaryotic expression vector pIRES2-EGFP.Recombinant bacterium fluid was sequenced by Shanghai Invitrogen Biotechnology Company,and named the correctly recombinant plasmid was pIRES2-EGFP-SNCG.After the identification by restriction endonuclease digestion and the nucleotide sequencing, the recombinant vector was stably transfected into human glial tumor U373 cell lines by G418. The expression of SNCG was detected by real-time RT-PCR and Immunofluorescence,cell cycle distribution was detected by FCM.Result:Verified by RT-PCR and pIRES2-EGFP vector cloning site primers on a bi-directional sequencing, DNA sequencing results confirmed that the recombinant plasmid insert sequence SNCG consistent with GenBank published sequences,It was proved that SNCG recombinant eukaryotic expression vector constructed correctly.The SNCG eukaryotic expression vector was constructed and verified. The U373/SNCG cell line model was established which stably transfected SNCG. After the recombinant plasmid pIRES2-EGFP-SNCG stably transfected U373 cells, used fluorescent microscope observing them in the same field, all the cells showed a visible view in the green fluorescence. The RT-PCR results showed that compared to U373 cells withou transfection,neo mRNA's expression in U373/neo and U373/SNCG cells were significantly increased; U373/SNCG cells SNCG mRNA expression level was significantly increased, and U373 cells without transfection has no difference between U373/neo cells.The relative fold change of SNCG in U373/SNCG cells was 17 compared with U373/neo cells. Meanwhile, Over-expressing SNCG decreased the blockage of cell mitosis which was induced by anti-microtubule drugs, with decrease in the G2/M phase 17%(P<0.05).Conclusion:The recombinant expression vector pIRES2-EGFP-SNCG has been constructed and stably transfected in the U373 cell lines successfully, initially confirmed the expression of SNCG had reduced anti-microtubule drug-induced U373 cell mitosis arrest,which lays a base for further study about the role of SNCG in glial tumor's invasion and metastasis in vitro.