CD146基因重组质粒的构建及蛋白表达

目的构建CD146原核、真核表达载体,证实融合蛋白在原核细胞的诱导表达以及在胃癌细胞内的表达和定位。方法提取人黑色素瘤细胞A875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,分别克隆至pCDNA3.1-myc/his以及pGEX-4T-3表达载体中。原核重组质粒鉴定后转入BL21细胞中并经了诱导表达及纯化,真核表达质粒转入胃癌MKN45细胞中,分别利用westernblot和激光共焦扫描显微技术检测了重组质粒的表达以及在胃癌细胞中的定位。结果 CD146全长基因序列克隆到原核、真核表达载体中,酶切鉴定片段为1930bp。原核诱导出了GST-CD146并进行了纯化,CD146在真核细胞中表达为113KD的糖蛋白,Westernblot检测到真核转染的myc/his-CD146表达,条带约为120KD,免疫荧光显示蛋白定位在胃癌MKN细胞膜。结论成功构建了CD146原核、真核表达载体,融合蛋白在胃癌MKN45细胞表达。

Construction of CD146 gene fusion plasmid and its recombinant protein expression

Objective To construct the recombinant plasmid of human CD146 gene and identify its recombinant protein expression in gastric cancer MKN45 cells.Methods Total RNA was extracted from A875 cells,CD146 coding sequence was amplified by polymerase chain reaction（PCR）method and cloned into pCDNA3.1-myc/his or pGEX-4T-3 vectors respectively.IPTG-induced GST-CD146 protein expression in BL21 cells and protein purification were identified by SDS-PAGE.The expression and localization of the pCDNA3.1-myc/his-CD146 recombinant plasmid in MKN45 cells were proved by western blot and immunofluorescence.Results CD146 was constructed into pGEX-4T-3,pCDNA3.1-myc/his successfully,the length of the fragment was about 1900 bp.The expression of IPTG induced GST-CD146 and protein purification were identified by SDS-PAGE and commassie blue staining.The myc/his-CD146 expression was identified in MKN45 cells by western blot with a molecular weight of about 120 Kda and localized in cell membrane.Conclusion The CD146 full length sequence was constructed successfully and cloned into prokaryotic and eukaryotic expressing vectors,and its fusion protein fusion protein was expressed in MKN45 cells.