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Construction and Identification of the hTfR Gene’s Plasmid and Its Expression in Hela Cell

Author: ZhangBin
Tutor: ZhouShunKe
School: Central South University
Course: Medical Imaging and Nuclear Medicine
Keywords: hela cell hTfR RT-PCR Molecular Imaging plasmid
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 18
Quote: 0
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Abstract


Objective Human transferrin receptor (hTfR) is a widely distributed transmembrane glycoprotein in the cell membrane, through specific binding proteins transferrin mediated iron transport.It also closely linked cell growth, proliferation and differentiation. It is low expression in normal mature cells, while overexpression of a variety of tumor cells. Combined with molecular probe technology makes hTfR could serve as ideal molecular imaging reporter gene in MR. In our reseach will use hTfR as MR report genes, production expression plasmid including coding region of hTfR, carry out detection and identified, provide the possbilility for the molecular imaging of MR in vivo and vitro.Method We purchase expression plasmid including full length of hTfR gene cDNA from Yr gene bank and reference Gene-Bank (No. BC001188) information for synthesis primers. Make coding region amplification of hTfR Through Reverse transcription polymerase chain reaction (RT-PCR) method and Building expression plasmid of pcDNA3.1 (+)-hTfR by digestion of enzyme and conjunction. After that, we do identification and sequencing. Using Lipofectamine to do transient transfection of Hela cell, made its overexpression of the protein of hTfR. Detect the expression of hTfR in anteroposterior period thourgh Westen-Blot and mRNA through RT-PCR, identify the efficiency of transfectionResults pOTB7-TFRC plasmid as a template, amplify the coding region of hTfR gene through Reverse transcription polymerase chain reaction (RT-PCR) constructed pCDNA3.1 (+)-hTfR overexpression plasmid successfully, identify the correctness through sequencing; RT-PCR showed that transfected pCDNA3.1 (+)-hTfR plasmid group than the control group in generated hTfR mRNA was significantly higher (P<0.05); Western-Bolt showed that the expression of TfR protein in the transfection of pCDNA3.1 (+)-hTfR cells is significantly higher than no transfection cell (P<0.05).Conclusion We construct pCDNA3.1 (+)-hTfR expression plasmid successfully, there are higher expression of hTfR protein and hTfR mRNA after transient transfection; establish infarctate foundation to empirical reseach of molecular imaging of MR.

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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