Construction Of Lentiviral Vector Carrying ENOS/F92A-Caveolin-1Gene And Functional Analysis

Background and ObjectivePulmonary arterial hypertension (PAH) is a progressive disease associatedwith a variety of causes. The pathological basis of PAH is the deformity ofpulmonary endothelium or vascular structure, which resulting in pulmonaryvascular endothelial dysfunction and pulmonary vascular remodeling. Therefore,to treat the PAH effectively, endothelial dysfunction and pulmonary vascularremodeling must be reversed. Endothelial nitric oxide synthase (eNOS), existingin endothelial cells mainly, is an important molecule for regulation of vasculartone and permeability. Nitric oxide (NO) is mainly produced by the endothelialcells of vascular wall. NO may play an important role in the process of anti-PAHthrough multiple pathways. Caveolin-1(Cav-1), which is located in Caveolae, isa key negative regulator of production of eNOS. Phenylalanine combinationwith eNOS play inhibitory effects.Phenylalanine located at92position ofCaveolin-1. But alanine (F92A) replaced phenylalanine and obtained mutantF92A-Caveolin-1(F92A-Cav-1, Caveolin-1mut),which doesn't inhibit the roleof eNOS.Activity of eNOS can be increased and the secretion of NO can beenhanced by changing of Caveolin-1gene, thus this method is conducive to thetreatment of PAH.This study is designed to construct lentiviral vectors carrying eNOSand F92A Caveolin-1(Caveolin-1mutation, mut Caveolin-1) as target gene. Thefunction of the lentiviral vectors will be tested and analysed. The constructionof lentiviral vectors will lay scientific foundation for further future research.Methods 1) Primers were designed according to plasmid (eNOS, F92A-Cav-1) andthe appropriate restriction sites in5' and3' ends were added,respectively. Therestriction sites is the same as the lentiviral vector backbone(pLVX-mCMV-mCherry). The target gene eNOS and F92A-Cav-1wereamplified by Polymerase chain reaction?PCR?methods; through the steps suchas digestion, ligation, transformation, clones were picked and positive cloneswere screened and positive clones were identified by PCR? sequencing andrestriction enzyme digestion, the corrected clones plasmid named aspLVX-eNOS-P2A-F92-Caveolin-1-mCMV-mCherry(LV-eNOS/F92-Cav-1).2)Lentiviral lenti-eNOS/F92-Cav-1can be obtained by lipofection method(Lipofectamine2000)and using human embryonic kidney cells (293-T cells) forpackaging. Viral supernatant was collected after48hours and72hours ofinfection and concentrated by PEG-it. Virus titer was determined by dilutionmethod and infected index (multiplicity of infection, MOI) was obtained.3)when MOI is one,293-T cells were infected by lentiviral particlesLenti-eNOS/F92-Cav-1, with a fluorescence microscope to detect transductionpositive rate.4? In order to detect the corresponding function of the constructedplasmid,293-T cells were divided into three groups, experimental group:Lentiviral Particles (Lenti-eNOS/F92-Cav-1) infection group; empty vector(pLVX-mCMV-mCherry) infection group; Control group.293-T cells ofexperimental group was infected when MOI=1, and throughimmunofluorescence chemical methods to detect eNOS protein (with GFP)expression. Then through DAF-FM DA method (NO fluorescent probe) to detectNO production.Results1.Positive clones were identified by PCR, sequencing and restrictionenzyme digestion, and the results suggest that lentiviral expression plasmidLV-eNOS/F92-Cav-1was constructed successfully.2.The virus titer reached to2×108TU/ml and viral infections Index (MOI)is one.3.The positive rate of infection of293-T cells were detected under afluorescence microscopy up to80%? when MOI is one.4.Both eNOS protein expression and NO production volume ofexperimental group increased, as compared with the negative control group and blank control group.Conclusions1.Lentivirus expression plasmid LV-eNOS/F92-Cav-1, carrying target geneeNOS/F92A-Caveolin-1, was constructed successfully.2.Through the detection of its function, both eNOS protein expression andNO production volume of experimental group increased. This result furtherlyexplained that the lentivirus vector lenti-eNOS/F92-Cav-1was constructedsuccessfully.