The Experimental Study On Construction Of Recombinant Adenovirus Vector With Human Protein Kinase C β₂ By AdMax System

PART ONE CLONING AND SEQUENCING OF HUMAN PKCβ2 GENE ORFObjective: To clone human PRKCB1 gene open reading frame in order to further research on function of human PKCβ2. Methods: With RT-nested PCR method,the amplified frame,from human umbilical vein endothelial cell(HUVEC), which include total length of human PRKCB1 ORF,was inserted into T vector after adding the A tail.With blue white screening, the gene ORF encoding human PKCβ2 was obtained with special primers from recombinant plasmids and linked into T vector.The plasmid was identified by sequencing.Results: The human PRKCB1 ORF was amplified successfully with RT-nested PCR and T/A cloning. The gene sequence was completely consistent with that reported in GenBank.Conclusions: Human PRKCB1 gene ORF was successfully cloned. The strategy of cloning may provide technical references for some genes hard to be cloned. PART TWO CONSTRUCTION AND TRANSFECTION OF EUKARYOTIC EXPRESSION VECTOR OF HUMAN PRKCB1 CONTAINING ENHANCED GREEN FLUORESCENCE PROTEIN GENEObjective: To construct the eukaryotic expression vector of human PRKCB1 containing enhanced green fluorescence protein gene and transfer into human umbilical vein endothelial cell(HUVEC).Methods: The pReceiver-M29-PRKCB1 eukaryotic expression plasmid was constructed by frame amplified from pMD18-T-PRKCB1 plasmid and pReceiver-M29 plasmid containing enhanced green fluorescence protein gene. Then the recombinant plasmids were identified by enzyme analysis and DNA sequencing. According to optimized conditions,the eukaryotic expression plasmids were transfered into HUVEC and observed under fluorescence microscope.After that, transfection efficiency was calculated under random vision.Results: The gene sequence was completely consistent with that reported in GenBank. The enhanced green fluorescence protein could be observed in HUVEC after 48 hours.Transfection efficiency was 18.62%.Conclusion: The pReceiver-M29-PRKCB1,an eukaryotic expression plasmid,is successfully constructed and transfered into HUVEC. It is the molecular instrument for screening HUVEC stably expressing human protein kinase cβ2 and isolating protein complex.PART THREE CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT ADENOVIRUS VECTOR WITH HUMAN PROTEIN KINASE Cβ2 BY CRE/LOXP SYSTEMObjective: To construct the recombinant adenovirus vector with human protein kinase cβ2 by Cre/loxP system,for further research on function of human PKCβ2.Methods: The PRKCB1 gene was amplified from pMD18-T-PRKCB1 plasmid and was directly cloned into shuttle plasmid pDC315 to construct shuttle plasmid.Then the combinant shuttle plasmid and adenovirus genomic plasmid pBHGlox△E1,3Cre were contransfected into 293 cells to construct recombinant adenovirus Ad-PRKCB1.The virus titer was calculated by TCID50.The Ad-PRKCB1 was verified by immunocytochemistry and RT-PCR. Results: Restrictive endonuclease digestion and PCR showed the gene was correctiy cloned into shuttle plasmid.The CPE was observed under inverted microscope through homologous recombination. The virus titer was 7.9×109IU/ml.The effective expression of human PKCβ2 identified by immunocytochemistry and RT-PCR.Conclusion: A recombinant adenovirus vector with human PKCβ2 is constructed by Cre/loxP system.