Experiment Study On Construction Of AdPEDF And It's Eukaryotic Expression

Objective: To construct recombinant adenovirus carrying pigmentepithelium-derived factor (PEDF) gene for the gene therapy research of choroidalneovascularization(CNV).Methods: (1)The PEDF cDNA was prepared by nest RT-PCR technique using thetemplate mRNA isolated from the tissue of the retina of the BN rat. The PEDFcoding sequence was cloned into the vector pGEM-Teasy and then sequenced. Apair of primers for PEDF were designed which contained EcoRâ… and Hindâ…¢specific sites on each end, then the PEDF coding sequence was subcloned into aadenovirus expression plasmid pDC316, then the recombinant vector pDC316/PEDFwas analysised by restricted endonuclease Sac I, and then sequenced. The 293 cellswere cotransfected with pDC316/PEDF and pBHGloxΔE1,3Cre by Lipofectamine2000, thus AdPEDF was constructed by Cre/loxp site intracellular homologousrecombination. After the recombinant adenovirus was identified by PCR, therecombinant adenovirus stock reproduced in 293 cells and was concentrated by Csclultracentrifugation, and detected the titer of the recombined virus. (2) Detecting theexpression of PEDF protein in eukaryotic cell. HepG2 cells were infected withAdPEDF in vitro. 1hours later, the medium was changed by culture medium withoutFBS, 48 hours later, the supernatant was collected, then detected the expression ofPEDF by Western blot.Results: (1) As compared with the sequences of the rat PEDF mRNA published inNCBI Sequence Viewer(NM-177927), the PEDF cDNA fragment contained all the sequences of open reading frame of the rat PEDF gene. The plasmids pDC316/PEDFwas constructed successfully, and the nucleotide sequences of pDC316/PEDF wereconfirmed by sequencing. The 293 cells were cotransfected with pDC316/PEDF andpBHGloxΔE1,3Cre, and thus AdPEDF were constructed by Cre/loxp siteintracellular homologous recombination. The recombinant adenovirus titer was up to3.08×10¹⁰ pfu/mL after amplification and Cscl gradient purification, which isqualified for experiments in vivo and in vitro. (2)Western blot confirmed that thePEDF protein was significantly expressed in the infected group, while was not incontrol group. It was demonstrated that AdPEDF could transfect eukaryotic cells, andthe PEDF protein can be detected in supernatant of the cells.Conclusion: The successful construction of AdPEDF lay an important foundation forgene therapy of CNV.