真核荧光蛋白表达载体pEGFP-C3-B7.2-MAGE-1的构建与表达

Construction and expression of eukaryotic fluorescent protein expression vector pEGFP-C3-B7.2-MAGE-1

目的:构建表达B7.2和MAGE-1的绿色荧光共表达载体pEGFP-C3-B7.2-MAGE-1,并在真核细胞中表达。方法:根据GenBank中的序列,对B7.2、MAGE-1各设计一对两端带有特定限制性酶切位点的引物,分别从乳腺组织、乳癌组织提取总RNA,进行RT-PCR后,将两扩增产物分别克隆在pGEM-T载体,经测序证实碱基序列无误后,双酶切pGEM-T载体,回收目的片段,将两目的片段亚克隆至真核绿色荧光蛋白共表达载体(pEGFP-C3)上,并转染真核细胞,观察其在真核细胞中表达。结果及结论:pEGFP-C3-B7.2-MAGE-1真核表达载体经酶切及基因序列分析验证,PCR扩增片段与选择的目的片段序列相符,pEGFP-C3-B7.2-MAGE-1真核表达载体构建成功。该表达载体转染EC9706细胞后,用免疫荧光显微镜观察和RT-PCR分析,该重组载体能够在真核细胞中广泛表达。

Aim ：To construct green fluorescent expression vector pEGFP-C3-B7.2-MAGE-1 and induce its expression in eukaryotic cells. Methods： According to the published sequence in GenBank, a pair of primers containing the sites of given restrictive endonuelease at both ends were designed and synthesized for B72 and MAGE-1 , respectively. Reverse transcriptional PCR （RT-PCR） of the total RNA extracted from breast tissue and breast-cancer tissue was performed, the products were cloned into pGEM-T vectors, respectively,after the amplified product was examined by sequence determination, digest these pGEM-T vectors with two restriction endonuclease, retrieval the target fragments, subclone both target fragments into the pEGFP-C3 fluorescent expression vectors. Then the expression of the eukaryotic cell which the expression vector was transfected into was observed. Results and conclusions ： The target fragment was obtained as expected. The restriction enzyme digestion and sequence analysis showed that recombined cloning expression vector pEGFP-C3-BT. 2- MAGE-1 had been constructed successfully. Then the transfected EC9706 cell was observed with immunofluorescence microscope and anlysize it by RT-PCR essay, finding that the recombination vector can generally express in eukaryotic cell.