Highly Efficient Co-expression Of VEGF₁₆₅ And FGF₂ Under The Control Of The Cardiac-specific MLC-2v Core Promoter And RTP801 Core Hypoxia-response Enhancer

Objective: To validate the enhancement property of the core hypoxiareactive enhancer of RTP801 promoter to the expression of VEGF₁₆₅and FGF₂ under CMV promoter,then furtherly validate the highlyefficient cardiac-specific and hypoxia-response co-expression ofVEGF₁₆₅ and FGF₂ under the control of the cardiac-specific MLC-2v corepromoter and RTP801 core hypoxia-response enhancer.Metheds:Acquire Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ fragment from the recombinantplasmid pSEC/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ by PCR(Igκis the DNAfragment of secretary targeting signal Igκ-chain leader),then switch itinto the multiple clone sites of the vector pIRES to construct therecombinant plasmid pIRES/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂.Acquire the337-511bp(plus amphi-restriction enzyme sites and protective basylscome to 202bp) of the core hypoxia-response enhancer of RTP801promoter Genebank NC000076) from mouse genome DNA by PCR,then use it to replace the CMV enhancer (150-390bp) of the recombinantplasmid pIRES/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ to construct therecombinant plasmid pIRES/RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂, and then transfect it to the 293 cells with biodegradable hyperbranched polyethylenimine (PEI) in vitro.The infected cells werecultivated for 36 hours under normal and anoxic conditionrespectively.The expression of VEGF₁₆₅ and FGF₂ in the cells weredetected by Western-blot,the expression of the secretary proteinVEGF₁₆₅ and FGF₂ in the cell culture fluid were detected byELISA.Aquire the 1714-1975bp 261bp core promoter (plusamphi-restriction enzyme sites and protective basyls come to 289bp)ofthe MLC-2v promoter (myosin light chain 2v promoter,GenebankU26708) from rat genome DNA by PCR, then use it to replace the CMVpromoter of the constructed recombinant plasmid pIRES/RTP801 coreHRE/Igκ-VEGF₁₆₅/IRES/ Igκ-FGF₂ to construct recombinant plasmidpIRES/MLC-2v core promoter/RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂,and then transfect it to the rat myocardial cell line H₉C₂ cellsand 293 cells with biodegrable hyperbranched polyethylenimine(PEI) invitro.The infected cells were cultivated for 12 hours under normal andanoxic condition respectively.The expression of VEGF₁₆₅ and FGF₂ inthe cells on the level of mRNA was detected by RT-PCR. Results: Theconstruction of eukaryotic expression vector pIRES/RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ was confirmed to be successful by PCRand restriction enzyme dissection analysis. The highly efficientco-expression of VEGF₁₆₅ and FGF₂ in the recombinant plasmidpIRES/RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ mediated by RTP801 HRE in the anoxic 293 cells was confirmed by the detection ofWestern-blot and ELISA. The construction of eukaryotic expressionvector pIRES/ MLC-2v core promoter /RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ was confirmed to be successful by PCRand restriction enzyme dissection analysis. The highly efficient andspecific co-expression of VEGF₁₆₅ and FGF₂ in the recombinant plasmidpIRES/MLC-2v core promoter/RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ mediated by MLC-2v core promoter and RTP801 core HREin the anoxic rat myocardial cell line H₉C₂ cells was confirmed by thedetection of RT-PCR and ELISA. Conclusion The acquired RTP801core hypoxia-response enhancer has the function of significantlystrengthening downstream gene expression under hypoxia condition,onthis base we construct the eukaryotic plasmid pIRES/RTP801 core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ and get the highly efficient expression ofVEGF₁₆₅ and FGF₂ under hypoxia. Cardiac-specific MLC-2v corepromoter and RTP801 core hypoxia-response enhancer has the functionof eahancing downstream gene cardiac-specific and hypoxia-responsehighly efficient expression when they are used together, on this base weconstruct the eukaryotic plasmid pIRES/MLC-2v core promoter/RTP801core HRE/Igκ-VEGF₁₆₅/IRES/Igκ-FGF₂ and it high efficiently expressesVEGF₁₆₅ and FGF₂ in anoxic rat myocardial cell line H₉C₂ cells. Thisexperiment may serve as groundwork for further research of constructing highly efficient vectors in gene therapy of ischemic disease above allischemic heart disease.