多发性骨髓瘤黏蛋白-1基因真核表达载体构建及其在COS-7细胞中的表达

Construction of Eukaryotic Expressing Vector of Multiple Myeloma Mucin-1 and Its Expression in COS-7 Cells In Vitro

本研究旨在构建多发性骨髓瘤黏蛋白1(两串联)基因(muc1-2vntr)的真核表达载体及该重组体在COS-7细胞中的表达,为研制多发性骨髓瘤基因疫苗奠定基础。以MUC1-2VNTR蛋白的编码基因作为目的基因,在其前端加上KOZAK序列后,两端分别加上HindⅢ和XbaⅠ酶的酶切位点,将人工合成的全基因定向插入pcDNA3.1/myc-his B载体中,转化E.coli感受态细胞获得重组菌株,经酶切及测序鉴定为pcDNA3.1-2vntr/myc-hisB重组质粒后,采用Lipofectimine脂质体介导法转染COS-7细胞,用荧光显微镜对荧光表达进行观察,然后用Western blot检测重组体在细胞内外的表达。结果表明:合成的MUC1-2VNTR基因全长约140bp,所构建的pcDNA3.1-2vntr/myc-hisB重组质粒经双酶切及测序鉴定后,与预期片段大小相符,并含有完整的阅读框架和目的基因。将其转入COS-7细胞后48小时,荧光显微镜和Westernblot检测均可证实黏蛋白1表达。结论:成功的构建了多发性骨髓瘤真核表达载体pcDNA3.1-2vntr/myc-hisB,并在COS-7细胞中成功地表达黏蛋白1,这为黏蛋白1的功能研究和多发性骨髓瘤基因疫苗的研制提供了实验基础。

In order to construct an eukaryotic expression vector for gene of multiple myeloma mucinl （mucl-2vntr） gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine, mucl-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind Ⅲ and Xba Ⅰ restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-bis B vector, and the resulted recombinant vector was transformed into E. coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3. 1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using mucl monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr /myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.