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Construction and identification of pIRES_2-AcGFP_1-Nuc-TFPI eukaryotic dual expression vector

Author: LouSha
Tutor: LengXiGang；SongCunXian；LiuTianJun
School: Peking Union Medical College , China
Course: Biomedical Engineering
Keywords: People TFPI GFP Gene therapy Vascular restenosis
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 27
Quote: 0
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Abstract


Human tissue factor pathway inhibitor (TFPI) body extrinsic coagulation pathway inhibitor , inhibit FXa and FVIIa-TF complex functions , the main one of the anti - clotting factors . TFPI both inhibition of thrombosis , smooth muscle cell proliferation and monocyte chemoattractant triple function , and therefore has a good prospect of application in the prevention and treatment of vascular restenosis . The experimental build of tissue factor pathway inhibitor factor (TFPI) eukaryotic expression vector that the TFPI gene therapy for the prevention of restenosis study provides an effective means . PIRES-TFPI In this experiment, containing the full-length cDNA as a template , PCR amplification of the the TFPI full-length cDNA inserted into pIRES2-AcGFP1-Nuc vector after digested build dual cistronic eukaryotic expression vector , recombinant plasmids were pIRES2-AcGFP1-Nuc-TFPI. enzyme mapping , PCR amplification and sequencing named after transfection of NIH 3T3 cells , respectively, using fluorescence microscopy , RT-PCR and ELISA method for detection of GFP or TFPI in the cell expression. Enzyme mapping cell memory detected in the expression of the GFP protein fluorescence microscope ; RT-PCR confirmed transferred by TFPI gene PCR amplification and DNA sequencing confirmed bicistronic eukaryotic expression vector pIRES2-AcGFP1-Nuc-TFPI constructed correctly ; TFPI mRNA expression in transfected cells increased ; ELISA detection showed that TFPI protein normally secreted into the extracellular TFPI gene transfected cell culture supernatant TFPI protein . In this study, the use of genetic engineering techniques , successfully constructed the eukaryotic expression vector including human tissue factor pathway inhibitor and green fluorescent protein , human tissue factor pathway inhibitor factor and green fluorescent protein gene from a double - Shun sub- mRNA mutual simultaneous expression without interference , to achieve the purpose of human tissue factor pathway inhibitor expression can be determined by observing the green fluorescent protein , and to ensure that the intracellular expression of human tissue factor pathway inhibitor can effectively secreted into the extracellular tissue factor pathway for human inhibiting factor for disease Control and Prevention study provides an effective research tool .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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