中文摘要：

目的 构建CD146真核表达载体,证实融合蛋白在骨髓瘤细胞内的表达、定位。方法 提取人工具细胞Hela细胞的总mRNA并进行反转。以反转录的cDNA为模板PCR扩增CD146全长编码基因,克隆至pCDNA3.1-3×Flag表达载体中。质粒转入骨髓癌NCI-H929细胞中,分别利用western blot和激光共焦扫描显微技术检测了重组质粒的表达以及在骨髓癌细胞中的定位。结果 CD146全长基因序列克隆真核表达载体中,酶切鉴定片段为1930bp。CD146在真核细胞中表达为113KD的糖蛋白,利用Western blot检测到真核转染的Flag-CD146表达,条带约为120KD,免疫荧光显示蛋白在细胞膜上有明显定位。结论 成功构建了CD146真核表达载体,并验证了其表达定位。

英文摘要：

Objective To construct the recombinant plasmids of human CD146 gene and identify its recombinant fusion protein expression in bone marrow cancer cells. Methods The total RNA was extracted from Hela ceils, CD146 coding sequence was amplified by polymerase chain reaction （PCR） and cloned into pCDNA3.1-Flag or pGEX- 4T-3 vectors respectively. The expression of IPTG-induced GST-CD146 protein in BL21 cells and protein purification were identified by SDS-PAGE. The expression and localization of pCDNA3.1-Flag-CD146 recombinant plasmid in NCI-H929 cells was proved by Western blot and immunofluorescence. Results CD146 was constructed into pCDNA3.1-Flag successfully, the length of the fragment was about 1900 bp. The expression of Flag-CD 146 in NCI-H929 cells was identified by Western blot with a molecular weight of about 120 KDa. Conclusion CD 146 full length sequence was successfully cloned into eukaryotic expressing vectors and expressed positively in NCI-H929 cells.