The Construction Of Adenovirus Vector Targeting Mouse Embryonic Fibroblasts And Its In Vitro Experiments

Mouse embryonic fibroblast(MEF)cells are one of the most common mammalian embryonic cells,which are isolated from the mouse embryonic and are adherent with a spindle form.Currently,MEF cells are extensively used in pluripotent stem cells(iPS)technique as one of the most common target cells with reprogramming factors Oct4?Sox2?c-Myc and Klf4 genes.The application of stem cells has bright prospects in clinical research area and has no ethical issues.Therefore,the procedure of induction of mouse fibroblasts into pluripotent stem cells has become a common approach in iPS research area.However,the mechanism of cell transformation is still unknown and transfection efficiency need to be further improved.The human adenovirus type5(Ad5)have no integration into the chromosome and low human pathogenicity.Aviod having any safety issue of the iPS in clinical application.Therefore,Ad5 vector is of great significance in the treatment of iPS applications.This vector infects the host cell through coxsackie-adenovirus receptor CAR(Coxsackie and adenovirus receptor)on the cell surface.It has become one of the most common and promising viral vectors in iPS.However,the CAR expression is very low in mouse embryonic fibroblast cells,resulting in low infection efficiency of Ad5 in these cells.One of strategies to solve this issue is to insert exogenous MEF cell-specific binding peptides into the adenovirus capsid protein.These exogenous peptides for modifying adenovirus genetically are mainly selected from the phage peptide library.In this project,a peptide with high affinity was selected by using phage display technology,which specifically binds to the mouse embryonic fibroblast cells from the phage library.The adenovirus with genetically modified capsid protein was able to infect the mouse embryonic fibroblast cells with a high efficiency.The main experimental methods and results of this study are as follows:(1)Biopanning of peptides from a random phage 12 peptides(PH.D.-12)library,which specifically binds to the mouse embryonic fibroblast cells:We utilized Based on the principle of aqueous-organic phase separation,we used biopanning and rapid analysis of selective interactive ligands(BRASIL)to select the specific MEF binding peptide,from a phage random 12 peptides library in mouse embryonic fibroblast cells,choosing NIH/3T3 as a negative cell.Twenty-three phage clones were randomly picked through 4 rounds biopanning.Then,seven different peptide sequences were analyzed by DNA sequencing,which include two high frequency peptides MetSHEMetNVHGYRN(the phage peptide named phage MSHE,the chemical synthesis of nucleotide sequence named MSHE)and DLWGYHYIDLDT(the phage peptide named phage DLWG,the chemical synthesis of nucleotide sequence named DLWG)VDLHYHPLKHKY(the phage peptide named phage VDLH,the chemical synthesis of nucleotide sequence named VDLH)and VDIPYHPLKHKY(the phage peptide named phage VDIP,the chemical synthesis of nucleotide sequence named VDIP),which have two common VD and YHPLKHKY motifs.There are three phage peptide only once.(2)Determination of the specific binding ability of 12 peptides phage clones(MSHE?DLWG?VDLH and VDIP)on the surface of MEF cells:By whole-cell enzyme-linked immunosorbent assay,it showed that the phage clone displayed MSHE?DLWG?VDLH and VDIP can bind to MEF cells.The results of the tittering assay and the cell immunofluorescence staining also indicated that these four phage clones can specifically bind to MEF cells.(3)Construction of adenovirus vector modified in Hexon of the capsid protein by GGGSMSHE?MSHE?DLWG?VDLH and VDIP peptides and detection of the infection efficiency:The GGGSMSHE?MSHE?DLWG?VDLH and VDIP gene sequences were synthesized chemically and cloned into the vector that is for rapid generation of capsid Hexon modified adenovirus through a one step ligation assay.To verify whether the linker of flexible peptides could reduce their interactions with the adenovirus vector,we inserted the GGGS flexible linker at the end of MSHE peptide and synthesized gene sequence GGGSMSHE.Successfully get five adenovirus vectors:pGHMAd5-GGGSMSHE?pGHMAd5-MSHE,pGHMAd5-DLWG?pGHMAd5-VDLH and pGHMAd5-VDIP.The constrcted vector was linearized by PacI,and then transfected into HEK293 cells with package adenovirus.The adenovirus were further amplified in HEK293 cells to get the sufficient virus stock,and then purified by the cesium chloride density gradient centrifugation and tittered.Four adenovirus(Ad5-eGFP-Hexon-GGGSMSHE?Ad5-eGFP-Hexon-MSHE?Ad5-eGFP-Hexon-VDLH and Ad5-eGFP-Hexon-VDIP)were identified at the final step.(4)Detection of the infection efficiency to MEF cells of the capsid modified adenovirus by GGGSMSHE?MSHE?VDLH and VDIP:The purified adenoviruses infected MEF and U20S at different titer for 48 h and the expression of reporter gene was observed and taken photo under a inverted fluorescence microscope.And the infection efficiency was quantified by a flow cytometry.The results showed that the modified capsid Hexon with GGGSMSHE and VDLH significantly improved the infection efficiency of adenovirus to MEF cell.(5)Construction of adenovirus vector with modified in Hexon of the capsid protein by GGGSMSHE,MSHE,VDLH and VDIP peptides and carried on iPS in E1.We totally constructed eight adenovirus vectors(pAd5/E1-Oct4/Hexon-GGGSMSHE?pAd5/E1-Sox2/Hexon-GGGSMSHE?pAd5/E1-c-Myc/Hexon-GGGSMSHE?pAd5/E1-Klf4/Hexon-GGGSMSHE?pAd5/E1-Oct4/Hexon-VDLH?pAd5/E1-Sox2/Hexon-VDLH?pAd5/E1-Klf4/Hexon-VDLH?pAd5/E1-c-Myc/Hexon-VDLH).In conclusion,our study generated 12 MEF binding peptides from phage library with modified the adenovirus capsid Hexon.The modified adenovirus by GGGSMSHE and VDLH peptides demonstrated a higher infection efficiency in MEF cell.Our data demonstrated that MEF cell is an ideal vitro model for iPS research.