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背景:腺病毒虽可携带骨诱导形成蛋白2基因转染靶细胞促进其分泌诱导形成蛋白2,但容易出现一些免疫应答反应,以质粒为载体的转染实验应具有良好的前景。目的:验证构建绿色荧光蛋白标记的人骨诱导形成蛋白2真核表达载体的可行性。设计:单一样本观察。单位:天津医院。材料:实验于2006-03/2007-03在天津医科大学卫生部激素与发育重点实验室(国家级)完成。含完整hBMP2基因片段的pcDNA3.1/CT-人骨诱导形成蛋白2质粒由李曦铭博士惠赠。双顺反子真核表达载体pSELECT-GFPzeo-MCS为Invivogen公司产品,Zeo购自Invivogen公司。pTA2-TEasy为鼎国生物技术有限公司产品,限制性内切酶BamHI和NheI、T4DNA连接酶(晶美生物工程有限公司),PCR上下游引物合成及测序(北京奥科生物技术有限责任公司)。方法:以重组质粒pcDNA3.1/CT-人骨诱导形成蛋白2为模板,结合已设计好的特异性引物,采用PCR方法亚克隆出人骨诱导形成蛋白2目的片段,将该片段分别与克隆载体pTA2-T-easy和双顺反子真核表达载体pSELECT-GFPzeo-MCS连接,转化入感受态DH5α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-人骨诱导形成蛋白2。主要观察指标:采用PCR法鉴定人骨诱导形成蛋白2序列,同时进行酶切及测序鉴定人骨诱导形成蛋白2是否克隆入pTA2-人骨诱导形成蛋白2重组质粒及真核表达载体pSELECT-GFPzeo-MCS中。结果:PCR获得长度约1216bp的目的片段,经与克隆载体pTA2-T-easy和真核表达载体pSELECT-GFPzeo-MCS连接,筛选及序列分析后,证实所插入目的片段与GenBank检索的BMP2cDNA序列(NM-001200)100%匹配。结论:成功构建含双顺反子绿色荧光蛋白标记人骨诱导形成蛋白2真核表达载体。
Background: Although adenovirus can transduce target gene into bone morphogenetic protein 2 (BMP-2) to induce its secretion and induce the formation of protein 2, some immune responses are easy to occur. Transfection experiments using plasmids as vectors should have good prospects. Objective: To verify the feasibility of constructing a green fluorescent protein-labeled human osteoinductive protein 2 eukaryotic expression vector. Design: Single sample observation. Unit: Tianjin Hospital. Materials: The experiment was performed at the State Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. The pcDNA3.1 / CT-human osteoinductive protein 2 plasmid containing the entire hBMP2 gene fragment was generously donated by Dr. Li Xi-Ming. The bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS is Invivogen and Zeo is from Invivogen. pTA2-TEasy is a member of GeneCopoeia Biotechnology, Restriction Enzymes BamHI and NheI, T4DNA ligase (Crystal Biotechnology), PCR primer synthesis and sequencing (Beijing Biosciences Biotechnology limited liability company) . Methods: Recombinant plasmid pcDNA3.1 / CT-human osteoinductive protein 2 was used as a template, combined with the designed specific primers, subcloned into human induced osteogenic protein 2 by PCR method, the fragment was cloned into the vector pTA2 -T-easy and bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS, and transformed into competent DH5α cells. The recombinant expression vector pSELECT-GFPzeo-human osteoinductive protein 2 containing green fluorescent protein was obtained by screening. MAIN OUTCOME MEASURES: Human osteoinductive protein 2 sequence was identified by PCR and digested with restriction endonuclease and sequenced to identify whether human BMP-2 was cloned into pTA2-human osteoinductive protein 2 recombinant plasmid and eukaryotic expression vector pSELECT-GFPzeo-MCS . Results: The target fragment of about 1216bp in length was obtained by PCR. After cloning vector pTA2-T-easy and eukaryotic expression vector pSELECT-GFPzeo-MCS, screening and sequence analysis confirmed that the inserted fragment was identical to the GenBank retrieved BMP2 cDNA sequence NM-001200) 100% match. Conclusion: The bicistronic green fluorescent protein (EGFP) -marked human osteoinductive protein 2 eukaryotic expression vector was successfully constructed.