hTfR基因载体的构建与鉴定及在Hela细胞中的表达

发布时间：2018-04-17 10:33

本文选题：hela细胞 + hTfR　；参考：《中南大学》2011年硕士论文

【摘要】：研究目的人类转铁蛋白受体(human Transferrin receptor, hTfR)是一种广泛分布于细胞膜的跨膜糖蛋白,通过与转铁蛋白的特异性结合来介导铁的转运,与细胞的生长、增殖、分化等有密切联系。它在大多数成熟的正常细胞中表达水平较低,而在多种肿瘤细胞中过量表达,结合分子探针技术,使得hTfR可以作为比较理想的MR分子影像报告基因。本研究运用hTfR作为MR报告基因,制作包含hTfR蛋白编码区的表达质粒,并对其进行检测鉴定,为下一步体外、体内MR分子影像研究打下基础。方法于Yr基因库中购买包含hTfR基因cDNA的表达质粒,参考Gene-Bank (No. BC001188)的信息合成引物,运用RT-PCR方法对hTfR基因的蛋白编码区进行扩增,经酶切、连接之后构建pcDNA3.1(+)-hTfR的表达质粒,经鉴定及测序正确后按脂质体转染方法瞬时转染Hela细胞,使该细胞过量表达hTfR。运用PCR及Westen-Blot的方法在基因水平和蛋白水平检测hTfR在转染前后的表达水平,鉴定转染效果。结果成功构建了pCDNA3.1(+)-hTfR过表达质粒,测序鉴定正确；通过脂质体法转染Hela细胞,RT-PCR显示转染pCDNA3.1(+)-hTfR质粒组较对照组生成的hTfR mRNA明显增高(P0.05)；Western-Bolt显示转染组较对照组编码的TfR蛋白明显增高(P0.05)。结论成功构建了pCDNA3.1(+)-hTfR表达质粒；质粒转染Hela细胞后表达的hTfR mRNA及hTfR蛋白均明显增高；为下一步分子影像学的实验奠定了基础。
[Abstract]:Objective Human transferrin receptor human Transferrin receptor (hTfR) is a transmembrane glycoprotein widely distributed in cell membrane. It is closely related to cell growth, proliferation and differentiation through specific binding with transferrin.The expression level of hTfR is low in most mature normal cells, but overexpressed in many kinds of tumor cells. Combined with molecular probe technique, hTfR can be used as an ideal molecular imaging reporter gene.In this study, hTfR was used as Mr reporter gene to produce expression plasmid containing coding region of hTfR protein, and it was detected and identified, which laid a foundation for further study of Mr molecular imaging in vitro and in vivo.Methods the expression plasmid containing hTfR gene cDNA was purchased from Yr gene bank.BC001188 primer was used to amplify the protein coding region of hTfR gene by RT-PCR method. After restriction endonuclease digestion, the expression plasmid of pcDNA3.1 (hTfR) was constructed. After identification and sequencing, Hela cells were transiently transfected by liposome transfection method.Overexpression of hTfR.PCR and Westen-Blot were used to detect the expression of hTfR before and after transfection at the gene and protein levels, and to evaluate the transfection effect.Results the pCDNA3.1 (-hTfR) overexpression plasmid was successfully constructed and identified by sequencing, and the pCDNA3.1 (pCDNA3.1- hTfR plasmid group) was transfected with Hela cells by reverse transcription-polymerase chain reaction (pCDNA3.1- hTfR plasmid group was significantly higher than the hTfR mRNA generated in the control group (P0.05) Western-Bolt display showed that the TfR protein encoded by the transfection group was significantly higher than that in the control group (P0.05).Conclusion the pCDNA3.1 (hTfR) expression plasmid was successfully constructed, and the expression of hTfR mRNA and hTfR protein increased significantly after transfection of pCDNA3.1hTfR into Hela cells, which laid a foundation for further molecular imaging experiments.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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