肿瘤转移关键酶-人乙酰肝素酶全长编码基因的克隆、表达及活性  

Cloning of full-length heparanase gene and expression of functional heparanase in mammalian cells

作　　者：李燕杰[1] 李吉学[1] 彭清忠[2] 汤国营[2] 林艳丽[2] 郭瑞锋[1] 朱厚础[2] 

机构地区：[1]郑州大学医学实验中心,河南郑州450052 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《第四军医大学学报》2005年第20期1881-1884,共4页Journal of the Fourth Military Medical University

摘　　要：目的:分离出乙酰肝素酶(heparanase,HPA)全长编码基因并在哺乳动物细胞中表达出有活性的HPA;探索此酶在哺乳动物细胞中的表达特性;并建立稳定表达该酶的细胞株.方法:通过PCR法从人胎盘cDNA文库中扩增HPA蛋白全长编码基因;构建真核表达载体,转化COS7细胞进行瞬时表达;分析HPA蛋白的活性和表达特性;转化入CHO细胞以筛选恒定表达该酶的细胞株.结果:分离扩增出1629bp的人HPA全长cDNA序列并构建了其真核表达载体;在转染后的COS7细胞和筛选出的CHO细胞裂解液中检测到较高的HPA活性和相对分子质量(Mr)大小约53×103的目的蛋白免疫印迹表达带.结论:本研究分离出人HPA编码基因全序列;在真核细胞内成功表达出有活性的人HPA,发现此酶在COS7和CHO细胞中表达在细胞内,很少分泌到细胞外;筛选出其恒定表达细胞株,为进一步研究HPA结构功能、开发其抗体及抑制剂提供了有力的工具.AIM： To explore the expression character of heparanase by isolating the full encoded gene and expressing the functional enzyme in mammalian cells. METHODS： We isolated the full encoded heparanase gene from human placenta cDNA library, cloned the gene into eukarytic expression vector pCDNA4/Myc-His, transfected it into COS-7 cells and CHO cells and then screened the CHO cells which permanently expressed heparanase in the selected medium. RESULTS： The activity of heparanase in the mammalian cell lysate detected was much higher than that in the culture medium. The molecular mass of the heparanase protein expressed in the transfected COS-7 and CHO cells was about 53 × 10^3 （Mr） by western blot. CONCLUSION： The full-length heparanase gene is isolated. The functional heparanase is expressed within COS-7 and CHO cells and few heparanse is secreted into medium. The positive CHO cells expressing active heparanase is screened, which will help explore its function and screen its antibody and antagonists from a variety of sources.

关 键 词：肝索裂合酶 基因表达 COS-7细胞 CHO细胞 

分 类 号：R73-3[医药卫生—肿瘤]