【摘要】 目的:构建信号肽-canstatin真核表达载体并在人食管癌细胞Eca-109中分泌表达。方法:利用点突变技术将小鼠纤溶酶原信号肽(SP)序列插入到载体pEGFP-C1 EGFP编码序列起始密码的后面,构建pEGFP-C1-SP载体。以pMD18T-Can为模板,扩增小鼠canstatin基因,构建信号肽-canstatin分泌型真核表达载体pEGFP-C1-SP-Can。用脂质体转染法瞬时转染人食管癌细胞Eca-109。利用W estern b lotting检测小鼠canstatin融合蛋白在Eca-109细胞中的分泌表达。结果:DNA测序证明所构建的带信号肽的中间载体pEGFP-C1-SP正确;酶切和DNA测序表明信号肽-canstatin分泌型真核表达载体pEGFP-C1-SP-Can构建正确,EGFP-cansta-tin融合蛋白在人食管癌细胞Eca-109中分泌表达。结论:获得了能分泌canstatin融合蛋白的真核表达载体,并分泌到人食管癌细胞Eca-109的细胞外,为小鼠canstatin的功能研究及应用于基因治疗提供了实验基础。更多还原

【Abstract】 AIM: To construct signal peptide-canstatin expression vector pEGFP-C1-SP-Can and express secretable mouse canstatin fusion protein in Eca-109 cells.METHODS: Site-directed mutagenesis was used in amplifying the signal peptide of murine plasminogen to construct the plasmid pEGFP-C1-SP.The cDNA of mouse canstatin,obtained from a cloning vector pMD18T-Can by PCR,was inserted into pEGFP-C1-SP to construct a secretable expression vector pEGFP-C1-SP-Can.Constructed plasmid pEGFP-C1-SP-Can was transiently transfected into Eca-109 cells via lipofectamine,and subsequently its secretable expression in the medium of cultured Eca-109 was observed by Western blotting.RESULTS: DNA sequencing and restriction enzyme analysis attested the validity of the constructed plasmids pEGFP-C1-SP and pEGFP-C1-SP-Can.EGFPcanstatin fusion protein was proved to be secretably expressed in Eca-109 by Western blotting.CONCLUSION: It is concluded that the constructed vector pEGFP-C1-SP-Can is valid and capable of expression in Eca-109,these findings provide a basis for testing the function of mouse canstatin and its application in gene therapy.更多还原