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目的:构建携EGFP的人SYNOVIOLIN基因真核表达载体。方法:应用基因重组技术,根据人SYNOVIOLIN基因序列和表达载体pIRES2-EGFP质粒上的多克隆位点设计引物,对含有SYNOVIOLIN基因的质粒pCDNA3-syno扩增,得到约1900bp的目的片段,进行T-A克隆。SalⅠ/BamHⅠ双酶切测序正确的重组质粒,回收SYNOVIOLINcDNA片段,将其亚克隆于pIRES2-EGFP载体的多克隆位点内得到质粒pIRES2-EGFP-syno。脂质体法转染HEK293细胞,用激光共聚焦显微镜和Western blot检测EGFP和SYNOVIOLIN在HEK293细胞的表达。结果:PCR,酶切及测序结果表明pIRES2-EGFP-syno真核表达载体构建成功,激光共聚焦显微镜和Western blot显示EGFP和SYNOVIOLIN蛋白在HEK293细胞中成功表达。结论:成功构建pIRES2-EGFP-syno真核表达载体并在HEK293细胞表达,为抗肌腱粘连的SYNOVIOLIN基因治疗研究奠定基础。
Objective: To construct eukaryotic expression vector of human SYNOVIOLIN gene carrying EGFP. Methods: The recombinant plasmid pCDNA3-syno containing SYNOVIOLIN gene was designed based on the human SYNOVIOLIN gene sequence and the multiple cloning site on the expression vector pIRES2-EGFP. The target fragment was about 1900bp and was cloned by TA cloning . SalI / BamH I double digestion sequencing of the correct recombinant plasmid, SYNOVIOLIN cDNA fragment was recovered, subcloned into the pIRES2-EGFP vector multiple cloning site plasmid pIRES2-EGFP-syno. HEK293 cells were transfected by lipofectamine. The expression of EGFP and SYNOVIOLIN in HEK293 cells was detected by confocal laser scanning microscopy and Western blot. Results: The eukaryotic expression vector pIRES2-EGFP-syno was successfully constructed by PCR, restriction enzyme digestion and sequencing. The expression of EGFP and SYNOVIOLIN protein was confirmed by confocal laser scanning microscopy and Western blot in HEK293 cells. Conclusion: The eukaryotic expression vector pIRES2-EGFP-syno was successfully constructed and expressed in HEK293 cells, which laid the foundation for the study of SYNOVIOLIN gene therapy for anti-tendon adhesion.