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目的构建晚期糖基化终末产物受体(RAGE)胞外段不同功能段V/VC1真核细胞表达载体,并在前列腺癌细胞株PC-3中表达,为进一步研究其在前列腺癌发病中的作用和机制打下基础。方法采用PCR方法扩增RAGE不同功能段V/VC1的序列,利用分子克隆技术将其重组于pcDNA3-HA载体中。利用PCR和测序鉴定克隆正确性。转染PC-3细胞,用Western blotting检测其表达,免疫荧光检测其在细胞中的定位。结果克隆的RAGE不同功能段V/VC1真核表达载体完全正确,Western blotting检测到RAGE不同功能段V/VC1的表达,免疫荧光检测其主要定位于细胞浆中。结论成功地构建了RAGE不同功能段V/VC1真核表达载体,其能够在PC-3细胞中表达。
OBJECTIVE: To construct a V / VC1 eukaryotic expression vector containing different functional segments of the extracellular domain of advanced glycation end-products receptor (RAGE), and to express it in prostate cancer cell line PC-3. To further investigate its role in the pathogenesis of prostate cancer The role and mechanism to lay the foundation. Methods The V / VC1 sequences of different functional segments of RAGE were amplified by PCR and cloned into pcDNA3-HA vector by molecular cloning technique. Clone correctness was identified by PCR and sequencing. The PC-3 cells were transfected and the expression of PC-3 cells was detected by Western blotting. The localization of PC-3 cells in the cells was detected by immunofluorescence. Results The V / VC1 eukaryotic expression vector of different cloned segments of RAGE was completely correct. The expression of V / VC1 in different functional segments of RAGE was detected by Western blotting. The immunofluorescence assay mainly located in the cytoplasm. Conclusion The V / VC1 eukaryotic expression vector with different functional segments of RAGE has been successfully constructed and can be expressed in PC-3 cells.