Construction And Identification Of Recombinant Adenovirus Eukaryotic Expression Vector Carrying Double Genes Namely BMP2and Human HIF1α^muand The Empirical Study Of Transfection The Adenovirus Into Rabbit MSCs

Objective1.To construct new recombinant adenovirus expression vector namelypAd-CMV-BMP2-IRES-HIF1α^muwhich can express in eucaryotic cell and takesalong human bone morphogenetic protein and hypoxia inducible factor1（HIF1α）interest protein meanwhile. Next to pack and change pAd-CMV-BMP2-IRES-HIF1α^muinto Ad-CMV-BMP2-IRES-HIF1α^muin HEK-293cells. meanwhile toconstruct Ad-CMV-BMP2-IRES-hrGFP-1, Ad-CMV-HIF1α^mu-IRES-hrGF，Ad-CMV-IRES-hrGFP-1as positive control vector and negative control vectorrespectively.2.To transfer double gene recombinant adenovirus eukaryotic expressionvector and positive control vector and negative control vector into rabbit marrowstromal cells （MSCs） with opti^mum^multiplicity of infection （MOI） and researchexpression quantity of all vectors. To angiogenesis of the effects ofdifferences between the experimental group and three Control groups atpromote generate bone and Blood vessels..To interpret for a new way of bonecoloboma disease at molecular level. MethodsPCR HIF1α^mu segments, BstXI and XbaI double enzyme cut andrecycling purpose extract. PIRES2-EGFP use BstXI and XbaI for microincisionenzyme cut. Recovery Larger pieces. Connect the recovery target gene withCarrier segment. Then introduced it into E. coli for amplification. PCR BMP2segments, NheI and BamHI double enzyme cut and recycling purpose extract.Connect the recovery target gene with Carrier segment. Then introduced it intoE. coli for amplification and Restructuring adenovirus expressing carrier. Usethe enzyme cut analysis、PCR for identified. The correct recombinant expressplasmid was transfect to HEK293cells and to definite information oftransfection involving hr GFP gene green fluorescence expression. To conductadenovirus packaging and to measure virus titre using endpoint dilution methodand at last construction of adenovirus expression vector called p Ad-CMV-BMP2-IRES-HIF1α^muis accomplished.The packaging of recombinant adenovirus vector includingAd-CMV-BMP2-IRES-hrGFP-1, Ad-CMV-HIF1α^mu, Ad-CMV-IRES-hrGFP-1which are positive control vector and negative control vector respectively isaccomplished. To select all adenovirus to transfer into pretreatment withdecaesadril rabbit MSCs following in order to detect BMP2and HIF1α genemRNA and protein expression with RT-PCR and Western blot.Results1. It shows that construction of pAd-HIF1α^mu-IRES-hrGFP-1is constructionand it is same to positive control vector and negative control virus vector.2It is observed found both the experimental group and the control groups^muchexpression of hrGFP and obvious cytopathic effect by fluorescence invertedmicroscope in HEK293A cells. The virus droplet degrees in turn were3.16×10⁸pfu/ml、1.3×10⁸pfu/ml、2.0×10⁸pfu/ml�'�1.9×108pfu/ml。3. It is found that there is no significance Statistical difference in HIF1α mRNA expression compare A and B or compare C、D and E group （P＞0.1）.but thereis significance statistic about quantity of HIF1α mRNA expression A group or Bgroup to C、D group or D group （P＜0.01） by RT-PCR decection after transfectinto rabbit MSCs.It is also found that there is significance statistic difference in BMP2mRNAexpression compare A and C group or compare B、 D and E group（P<0.05）.and there is significance statistic difference in BMP2mRNAexpression compare A and C group to B、D or E group （P＜0.01） by RT-PCR4. there is significance statistic about quantity of BMP2protein expressioncompared A、C group to B、D group or E group（P＜0.01） by Western blotdecoctions after transfect into rabbit MSCs. there is significance statistic aboutquantity of HIF1α protein expression compared A、B group to C、D group or Egroup（P＜0.01） by Western blot decoctions after transfect into rabbit MSCs.Conclusions1. Ad-CMV-HIF1α^mu-IRES-hrGFP-1was constructed successfully and so arethe control groups.2According to the RT-PCR and Western blot, The experimental group arebetter than the control groups in repair of bone defect and angiogenesis atmolecular level.