The Cloning Recombinant Retroviral Vector Construction And Cellular Expression Of Human CD9 Gene

CD9 expressed in a wide variety of cells,such as bone marrow, brain, and muscle cells. CD9 commonly expressed on several types of stem cells.It also expressed on oocytes and plays an important role in fertilization.CD9 is an integral membrane protein belonging to a family of tetraspan-membrane proteins (TM4) and is reported to play a role in cell adhesion, cell motility, and tumor cell metastasis.Therefore,we tried to clone the complementlary DNA of CD9 gene by RT-PCR and expressed it in mammal cells,for the purpose of more study about human CD9 protein.In this study,retroviral vector pBabe puro was used as expression vector because of its high infection efficiency and stable expression of outside gene. Then, the cDNA of CD9 amplified by RT-PCR from ES cell was ligated to vector. The pseudovirion produced in 293GP packaging cell that was transfected by the recombinant retroviral vector was used to infect SP2/0 cell in vitro.1 .The cDNA of human CD9 synthesis by RT-PCRHuman ES cell was cultured in DMEM till the number of cells reached 107,then total RNA was extracted using Trizol and quantified by uv spectrophotometer.Then proceeded RT-PCR, the PCR product was detected by electrophoresis in l% sepharose.The result showed that we got the exact gene.2 .Ligation of the cDNA of CD9 to T-vectorRT-PCR product was purified by PCR product purification kit,and thereafter ligated to pGEM-T vector.Then the recombination plasmid was transformed into E.coli DH5α,and the positive clones were selected,from which we extracted recombination plasmid and verified it by sequencing.3 .Construction of retroviral vectorT-CD9 recombination plasmid and retroviral vector pBabe puro were digested with restriction endonuclease and CD9 cDNA fragment recycled from Gel was linked using T4 DNA ligase to linearitied pBabe puro. The recombination plasmid was transformed into E.coli DH5α,and the plasmid in positive clones was extracted and the exact pBabe puro-CD9 were obtained by digesting with restriction endonuclease. 4 .Determination of recombinant retrovirus'pakageAfter 293GP cells were cultured in DMEM culture medium till to 60%-70% syncretizing. Recombinant plasmid(pBabe puro-CD9) and plasmid pVSVG were transfected into 293GP cell by calcium phosphate-DNA coprecipitation. Forty-eight hours later,the culture medium containing pseudovirion from the positive clones were collected and transfected it into SP2/0 cell5 . Expression of CD9 gene in recombinant SP2/0 cellsBy RT-PCR ,Westen blot and FACS verifying we conclude that CD9 gene was expressed in recombinant SP2/0 cells.