信号肽-canstatin真核表达载体的构建及其在Eca-109细胞中的分泌表达  

Construction of signal peptide-canstatin expression vector and its secretable expression in Eca-109 cells

作　　者：侯卫红[1] 田芳[1] 王建民[1] 王志超[1] 陈华艳[1] 薛乐勋[1] 

机构地区：[1]郑州大学医学院细胞生物学研究室,河南郑州450052

出　　处：《中国病理生理杂志》2006年第10期1935-1938,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金项目(No.30270031);河南省重大科技攻关项目(No.0122032500);河南省医学科技创新人才基金资助项目(No.2001006)

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。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.

关 键 词：CANSTATIN 血管生成抑制剂 食管肿瘤 ECA-109细胞 

分 类 号：R363[医药卫生—病理学]