title: Construction and characterization of a PDCD5 recombinant lentivirus             vector and its expression in tumor cells.
abstract: The function and mechanism of the programmed cell death 5 (PDCD5) gene is not completely understood, so it is necessary to build a stable and efficient PDCD5 recombinant lentiviral expression vector. The coding region of the PDCD5 gene was PCR amplified from pCMV-SPORT6. Next, the PDCD5 fragment and the pGC-FU vector were digested with the restriction enzyme AgeI and ligated by in-Fusion technology to build the pGC-FU-PDCD5 plasmid. Competent E. coli DH5α cells were transformed and positive clones were identified by PCR. The PCR products were digested and sequenced. After sequencing, positive clones were selected to grow and propagate. The pGC-FU-PDCD5 plasmid DNA was purified and mixed with the pHelper1.0 and pHelper2.0 packaging plasmids. They were co-transfected into 293T cells. The viral titer was measured by real-time PCR. The expression of GFP and PDCD5 was detected by both Western blotting and fluorescence microscopy. Additionally, the A498, ACHN, HCT116 and LoVo tumor cell lines were transfected with the virus supernatant, and PDCD5 expression was detected in these cells. The constructed pGC-FU-PDCD5 plasmid contained the correct PDCD5 gene sequence. Strong green fluorescence in both the cytoplasm and cell membrane was observed following transfection with purified pGC-FU-PDCD5 into 293T cells. The transfection rate was greater than 95%, and the expression of the PDCD5-GFP fusion protein was confirmed by Western blotting. The titer of the recombinant PDCD5 lentiviral condensed supernatant was measured to be 2 x 10(9) Tu/ml. The transfection efficiencies of A498, ACHN and HCT116 cells were greater than 90%. However, transfection efficiency of LoVo cells was lower but still greater than 70%. The PDCD5-GFP fusion protein was stable in these transfected tumor cells, as detected by Western blotting. In conclusion, a PDCD5 recombinant lentiviral vector was successfully constructed, and high expression of the plasmid was observed in tumor cells.
authors.affiliation: Department of Biotherapy and Laboratory of Biotherapy, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, PR China. sywang66@yahoo.com
authors.forename: Shiyong
authors.initials: S
authors.lastname: Wang
authors.forename: Xue
authors.initials: X
authors.lastname: Zeng
authors.forename: Yanping
authors.initials: Y
authors.lastname: Liu
authors.forename: Chunyan
authors.initials: C
authors.lastname: Liang
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Weili
authors.initials: W
authors.lastname: Du
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection; D044366:Transition Temperature; D019562:Viral Load; D019065:Virus Assembly
chemical_list: D051017:Apoptosis Regulatory Proteins; D009363:Neoplasm Proteins; C117283:PDCD5 protein, human; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins