中文摘要：

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-C1EGFP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-Cl-SP-Can。用脂质体转染法瞬时转染人食管癌细胞Eca-109。利用Western blotting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-Cl-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-Cl-SP-Can构建正确，EGFP-canstnstatin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。

英文摘要：

AIM： To construct signal peptide- canstatin expression vector pEGFP- Cl -SP- Can and express secretable mouse canstatln fusion protein in Eca- 109 cells. METHODS： Site -directed mutagenesis was used in amplifying the signal peptide of murine plasminogen to eonstrnct the plasmid pEGFP -Cl -SP. The cDNA of mouse canstatin, ob- tained from a cloning vector pMD18T - Can by PCR, was inserted into pEGFP - Cl - SP to construct a secretable expression vector pEGFP - Cl - SP - Can. Constructed plasmid pEGFP - Cl - 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 pEG- FP - Cl - SP and pEGFP - Cl - SP - Can. EGFP - canstatin fusion protein was proved to be secretably expressed in Eca - 109 by Western blotting. CONCLUSION： h is concluded that the constructed vector pEGFP- Cl -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.