膜表达热休克蛋白70真核表达载体的构建及其对肿瘤细胞的转染

The construction of vector expressing membrane-bound heat shock protein 70 in eukaryotes and transfection into melanoma cells

目的构建普遍适合性的膜表达热休克蛋白70(HSP70)的真核表达载体,修饰并建立HSP70表达并结合在细胞膜表面的肿瘤细胞。方法RT-PCR法从人胚肾中获取HSP70的cDNA,扩增成带酶切位点的目的基因。选择载体pDisplay,将目的基因插入载体多克隆酶位点中。DNA测序证实后,以脂质体转染的方法,将载体转染到黑色素瘤细胞。用G418抗性筛选获得阳性克隆。结果酶切电泳和DNA测序证实载体构建正确;目的基因的上游带信号肽序列,下游带跨膜序列。RT-PCR产物电泳、WesternBlotting、激光共聚焦显微镜和流式细胞术证实大量HSP70表达并结合在转染的黑色素瘤细胞膜表面。结论成功地构建了膜表达HSP70的真核表达载体;转染到黑色素瘤细胞后,细胞膜表面表达丰富的HSP70。转染细胞可以进一步制备新型瘤苗。

Objective To construct the vector expressing membrane-bound heat shock protein 70 (HSP70) and to transfect it to melanoma cells. Methods The HSP70 cDNA was obtained by RT-PCR of mRNA isolated from human embryo renal cells in reverse transcription. The PCR primers, including Sac Ⅱ and Sal Ⅰrestriction endonuclease sites, were designed. pDisplay, with signal peptide and PDGFR transmembrane sequence lying respectively at the two flanks of the multiple cloning site, was used to construct plasmid expressing membrane-bound HSP70 (mbHSP70) in eukaryotes and the DNA was inserted into the polyclone endonuclease sites in pDisplay. The construct pDisplay-mbHSP70 was verified by sequencing and transduced into melanoma cells. Result DNA sequencing showed that the vector expressing mbHSP70 in eukaryotes was correctly constructed; Electrophoresis for RT-PCR products, Western blotting, laser confocal microscopy and flow cytometry indicated that the melanoma cells expressing the protein onto the cell surface. Conclusion The vector expressing mbHSP70 in eukaryotes was successfully constructed; the melanoma cells expressing the protein onto the cell surface was verified.