携RSKA/hIGF-1杂交基因重组质粒的构建及表达

Construction of recombinant plasmid PBS-RSKA/hIGF-1 and its expression

背景:骨骼肌肌萎缩的治疗一直是基础和临床研究的难点和热点之一,近年来基因治疗在该领域得到了广泛的研究和关注。目的:拟构建携带大鼠骨骼肌α-肌动蛋白/人胰岛素样生长因子1(rat skeletal α-actin/human insulin-like growth factor-1,RSKA/hIGF-1)杂交基因的重组真核表达质粒,并观察其在C2C12细胞内表达。方法:登录Genbank数据库,查找RSKA启动子序列、hIGF-1cDNA序列和人生长激素3′尾端非编码区序列(3′UTR),把3段基因序列拼接后进行全基因合成,再将合成的全基因融合到PbluescriptⅡSK(+)[PBS]质粒上。用酶切电泳及测序检查质粒重组后序列的正确性;重组质粒转染C2C12细胞,应用RT-PCR和Western blot法分别鉴定转染细胞中hIGF-1 mRNA和蛋白的表达。结果与结论:重组质粒PBS-RSKA/hIGF-1酶切图谱与预期相同,测序验证插入片段全序列无改变。转染C2C12细胞后,RT-PCR及Western blot结果显示有特异条带出现,且差异明显。结果证明成功构建携RSKA/hIGF-1杂交基因的重组质粒,并在C2C12细胞中正确表达。

BACKGROUND：The treatment for skeletal muscle atrophy is a difficult and hot point both in basic and clinical researches.In recent years,gene therapy has aroused extensive attention.OBJECTIVE：To construct a recombinant plasmid for expressing the rat skeletal α-actin/human insulin-like growth factor-1（RSKA/hIGF-1） gene and to observe its expression in C2C12 cells.METHODS：The construct consists of rat skeletal a-actin promoter sequence,hIGF-1 coding sequence and human growth hormone 3＇UTR region.The construct was initially cloned into a pBluescript Ⅱ SK（＋） after synthesized,which contains the plasmid origin of replication and Ampicillin resistance gene.The sequence of synthesized hIGF-1 was confirmed by restriction analysis and DNA sequencing,then transfected into C2C12 cell.HIGF-1 gene was detected by RT-PCR and hIGF-1 was found by Western blot.RESULTS AND CONCLUSION：The restriction map of PBS-RSKA/hIGF-1 was identical to expectation,and sequencing verified that the inserted fragment was not changed.Specific bands could be seen in transfected C2C12 cells by RT-PCR and Western blot.Recombinant plasmid PBS-RSKA/hIGF-1 constructed successfully and it can be effectively expressed after being transfected into C2C12 cells in vitro.