可体内、外示踪的BMP2真核表达载体的构建和表达  

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenefic protein 2 eukaryotic expression vector

作　　者：黄洪超[1] 苗军[1] 刘春蓉[2] 胡永成[1] 夏群[1] 石可松[1] 

机构地区：[1]天津医院脊柱外科,300211 [2]武警医学院病理教研室

出　　处：《中华骨科杂志》2010年第12期1228-1234,共7页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金重点项目（30500516）

摘　　要：目的拟构建双顺反子增强型绿色荧光蛋白标记的人骨形态发生蛋白2fbonemorpho—geneticprotein-2，BMP2）真核表达载体，为其在真核细胞体内外示踪定位打下基础。方法以重组质粒pcDNA3．1／CT—hBMP2为模板，结合已设计好的特异性引物，采用PCR方法获得BMP2片段，将其分别与克隆载体pTA2-T-easy和真核表达载体pSELECT-GFPzeo—MCS连接，转化入感受态DH5（x细胞中，通过筛选得到含有绿色荧光蛋白的重组表达载体pSELECT—GFPzeo—hBMP2，并进行测序鉴定。将质粒转染CHO细胞后WesternBlot检测BMP2蛋白表达，用RT—PCR检测BMP2mRNA表达。结果琼脂糖电泳显示PCR产物为一条长约1216bp的带，重组表达载体pSELECT-GFPzeo—hBMP2经双酶切可得到约1216bp的片段，测序证实与Genebank中hBMP2序列完全相符，重组表达载体脂质体法转染CHO细胞48-72h后荧光显微镜下证实转染成功，经RT—PCR证实CHO细胞中存在BMP2基因表达，采用免疫荧光化学方法证实载体中CHO细胞表达BMP2蛋白。经WesternBlotting鉴定重组蛋白为分泌蛋白，相对分子质量在17×10^3左右。结论成功构建可体内外示踪增强含双顺反子绿色荧光蛋白人BMP2真核表达载体。Objective To construct green fluorescent protein （GFP）-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector. Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid peDNA3.1/CT-hBMP2 by using polymerase chain reaction （PCR）. hBMP2 gene was inserted into pTA2-T-easy and pSELECT-GFPzeo-MCS eukaryotie expression vector, and then transferred into competence DH5α ceils. After screening, pSELEC-GFPzeo-hBMP2 was obtained and identified by sequence analysis. The recombinant vector pSELECT-GFP zeo-rhBMP2 was transfected into CHO cells. The successful trasfection was verified by fluorescence microscope in 48-72 hours. The RT-PCR and immunofluorescence was used to confirm the hBMP2 expression. Western Blotting was used to detect the secretion of hBMP2. Results A 1216 bp fragment was obtained by PCR, the same as expectant fragment. The recombined pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was identified by restriction mapping and sequence analysis. The results were identical with that of reported hBMP2 sequence （Genebank NM-001200）. The successful transfection was verified by fluorescence microscope in 48-72 hours. The stable expression in eukaryotic cells was confirmed by immunofluorescence and RT-PCR which showed an obvious band between 1000-2000 bp. Western Blotting identified the immunogenicity of recombinant human BMP2 with the molecular weight of about 17-10s. Conclusion The pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was constructed successfully.

关 键 词：骨形态发生蛋白质类 蛋白质工程 CHO细胞 荧光抗体技术 间接 脂质体 

分 类 号：R686[医药卫生—骨科学]