Constuction Of HHCN4Expression Vector By Adenovirus And It Transfect HEK293A

Objective⑴To construct the adenovirus mediated herpolarization-activatedcyclicnueleotide-gated （HCN4） expression vector, and packing intorestructuringadenovirus AV-hHCN4-IRES/eGFP in HEK293A. Also build itsnegative control carrier AV-IRES/eGFP.⑵Transfering recombinant adenovirus expression vector and negativecontrol vector into HEK293A with multiplicity of infection （MOI） and researchthe expression in HEK293A. For biological pacing technology applicationestablished molecular experimental base.MethodsDue to the difficulty of amplification of full-length hHCN4, piecewiseconnection way of constructing entry vector pDown-hHCN4-IRES/eGFP.PDown-hHCN4-IRES/eGFP and adenovirus plasmid pAV.Des1d conduct LRreaction use Gateway Technology, construction of adenovirus vectorpAV-hHCN4-IRES/eGFP. By restriction enzyme analysis, PCR and sequencingfor identification. After purification using Lipofectamine2000mediatedtransfection of HEK293A, through the green fluorescent protein detection oftransfection, completion of the viral packaging, the successful construction ofthe recombinant adenovirus AV-hHCN4-IRES/eGFP and AV-IRES/eGFPnegative control adenovirus. Then using endpoint dilution method for thedetermination of virus titer.The two kinds of recombinant adenovirus transfect to HEK293A with the multiplicity of infection（MOI）.Western blot method for thedetection of cells transfected by hHCN4gene protein expression.Results⑴Identification of adenovirus vector containing pAV-hHCN4-IRES/eGFP:restriction enzyme identification, vector by PacI cut out2082bp and37334bpbands; recombinant adenovirus vector in the sequence of hHCN4andGenebank in hHCN4（NM₀05477.2） CDS perfectly, insert the correct direction.At the same time construction of the negative control group pAV-IRES/eGFP.⑵Packaging： Under inverted microscope fluorescence was observed inexperimental group which adenovirus transfected HEK293A cells and thenegative control group,within a large number of green fluorescent protein andhas obvious CPE.Tips for adenovirus packaging successful, virus titersdetected respectively1.26×108pfu/ml、1.15×108pfu/ml.⑶Western blot results: Protein expression in the experimental group wassignificantly higher than the negative control group，which has statisticallysignificant （P <0.01）.Conclusions⑴Ad-hHCN4-IRES/eGFP was constructed successfully and so is thenegative control group Ad-hHCN4-IRES/eGFP.⑵Successful packaging adenovirus AV-hHCN4-IRES/eGFP,AV-IRES/eGFP, and to detect the virus titer.⑶hHCN4gene protein express successfully in HEK293A.