人SYNOVIOLIN基因真核表达载体的构建及表达

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1900 bp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SYNOVIOLIN cDNA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。

Construction and expression of eukaryotic expression vector of human SYNOVIOLIN gene

Objective: To construct eukaryotic expression vector containing the EGFP-SYNOVIOLIN gene. Methods: According to the sequence of SYNOVIOLIN gene and the multiple clone sites of the expression vector pIRES2-EGFP plasmid, a specific pair of primers were designed and synthesize．PCR amplification of SYNOVIOLIN gene from the pCDNA3-syno plasmid was performed, and an approximate 1900bp objective fragment was achieved．The fragment was then cloned into T-A vector. The recombined vector confirmed by sequencing was digested by SalⅠ/BamHⅠ to obtain SYNOVIOLIN cDNA fragment, then the fragment was subcloned into the multiple sites of pIRES2-EGFP vector. The recombinant plasmid pIRES2-EGFP-syno was transfected into HEK293 cell by lipofectamine 2000. The result was examined using confocal microscopy. The expression of SYNOVIOLIN was detected by Western blotting. Results: PCR, DNA sequencing and restriction enzyme digestion analysis indicated that the eukaryotic expression vector pIRES2-EGFP-syno was constructed successfully. The expression of EGFP can be seen under confocal microscopy. Western blotting proved the protein expression of SYNOVIOLIN in HEK 293 cells. Conclusions: The SYNOVIOLIN cDNA is acquired and the eukaryotic expression vector, pIRES2-EGFP-syno is successfully constructed with efficient expression in HEK293 cells, which provide a good experimental basis for further study on the gene therapy of anti-tendon adhesion.