Construction Of PTie2-apoE₃-EGFP-N1Eukaryotic Expression Plasmid And Its Expression In Human Umbilical Vein Endothelial Cells

Objective: To construct a Tie2gene promoter regμLation of eukaryoticexpression vector pTie2-apoE3-EGFP-N1which is under the control of Tie2promoter and is to investigate the expression activity of the apoE3gene inhuman umbilical vein endothelial cells (HUVEC) with its effects on the host.Method: The first step is to get apoE3gene fragments from therecombination vector pEasyT1-apoE3by polymerase chain reaction(PCR), thenthe DNA fragment which contains the5′upstream Tie2gene eoding regionwere isolated from human peripheral blood genome. The above snippets weresequentially subcloned into the eukaryotic expression vector pEGFP-Nl. Afterconfirming the correct of recombinants,they were transformed into DH5αbacteria in order to get a lot of recombinants pTie2-apoE3-EGFP-N1. Therecombinants were transfected into HUVEC (human umbilical vein endothelial cells). Success or failure will be judged by observing the host cells in theexpression of green fluorescent protein (GFP).Results: It was successfully constructed the recombinant of expressionvector containing Tie2promoter and apoE3confirming by PCR,restrictionendonucleas, and sequencing.Conclusion:The recombinant eukaryotic expression vector wassuccessfully constructed,driven by the promoter of Tie2and producing theprotein apoE3. Objective: To construct apoE3gene promoter regμLation of prokaryoticexpression vector pET32a(+)–apoE3and is to investigate the expression activityof the apoE3gene in bacilli Bl21-DE3; To construct apoE expression profileof mouse.Method:The first step is to get apoE3gene fragments from the humantissue RNA by reverse transcript polymerase chain reaction(RT-PCR). Thenthe above snippet were subcloned into the prokaryotic expression vectorpET32a(+). After confirming the correct of recombinants,they weretransformed into DH5α bacteria in order to get a lot of recombinants pET32a(+)-apoE3. The recombinants were transfected into bacilli Bl21-DE3. Success orfailure will be judged by observing. At last,the protein produced by bacilliBl21-DE3with human apoE gene can be observed by the SDS-PAGE. Meanwhile, RNA of17kinds of mouse tissues,such as liver,heart and brain,was extracted and apoE gene expression profile could be analyzed by RT–PCR.Results:It was successfμLly constructed the recombinant of expressionvector containing apoE3confirming by PCR,restriction endonucleas, andsequencing. Mean while,in the tissue expression profile of mice, the strongestexpression of apoE gene was detected in liver tissue, followed by brain, spinalcord, heart, lung, spleen, kidney, stomach, small intestine, thymus gland, adrenalglands, pancreas, bladder, uterus, and arterial blood whose had different degreesof expression, but almost no expression in abdomen.Conclusion:The recombinant prokaryotic expression vector wassuccessfμLly construeted,inserted into pET32a(+) and producing the proteinapoE3. The tissue-specific expression in the profile of the mouse apoE gene issimilar to those of human and pig.