摘要
目的 构建人骨形成蛋白 - 2 (BMP- 2 )和骨保护素 (OPG)的共表达真核载体 ,研究其在小鼠成肌细胞C2 C12中的表达。 方法 以人骨肉瘤细胞系 MG6 3的总 RNA为模板 ,RT- PCR法获得人 OPG的编码区 c DNA,克隆入真核表达载体 p IRES2 - EGFP的多克隆位点 ,再将人 BMP- 2的编码区 c DNA克隆入 p IRES2 - EGFP中的 Bst X 位点 ,构建 BMP- 2和 OPG的双顺反子真核表达载体 p IRES2 - BMP- 2 - OPG,在阳离子脂质体介导下转染 C2 C12细胞 ,Western blot法检测 BMP- 2和 OPG的表达。 结果 1获得人 OPG编码区全长 c DNA。 2构建人 BMP- 2和 OPG的双顺反子真核表达载体 p IRES2 - BMP- 2 - OPG。 3经 Western blot检测 ,p IRES2 - BMP- 2 - OPG转染 C2 C12细胞后 ,细胞可稳定表达 BMP- 2和 OPG。 结论 构建了人 BMP- 2和 OPG的共表达真核载体 ,并可在 C2 C12细胞中稳定表达 ,为应用 BMP- 2和 OPG进行骨质疏松等疾病的治疗研究奠定了基础。
Objective To construct a co expressing vector of human bone morphogenetic protein 2 (BMP 2) and osteoprotegerin (OPG) and to determine the expression of BMP 2 and OPG in myoblast C2C12. Methods Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription polymerase chain reaction (RT PCT) method and cloned into sites EcoR Ⅰ and Bam HⅠ of mammalian expressing vector pIRES2 EGFP, and the cDNA encoding region of human BMP 2 was cloned into endonucleases site BstX Ⅰ. Then the recombinant plasmid pIRES2 BMP 2 OPG was transformed into C2C12 cell line, the expression of OPG and BMP 2 were determined by Western blot assay. Results The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2 BMP 2 OPG was constructed successfully. Human OPG and BMP 2 co expression cell line C2C12 was selected and confirmed by Western blot analysis. Conclusion The co expressing vector of OPG and BMP 2 is constructed and can expressed stably in myoblast C2C12. The co expression of human OPG and BMP 2 may be logical approach for treatment of osteoporosis and bone metastasis.
出处
《中国修复重建外科杂志》
CAS
CSCD
2003年第1期1-4,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目 (39870 792 )
