人骨肉瘤细胞株中骨形态发生蛋白-1的检测、克隆和表达  

Detection, cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines

作　　者：陈冬瑛[1] 朱全胜[1] 刘超[2] 丘钜世[1] 

机构地区：[1]中山大学中山医学院病理学教研室,广东广州510089 [2]广州市公安局DNA鉴定中心,广东广州510000

出　　处：《中国病理生理杂志》2003年第6期751-756,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目 (No .39770 30 1)

摘　　要：目的 :利用基因工程技术生产人骨形态发生蛋白 - 1的高保守区多肽 ,为进一步产生广谱型BMP - 1抗体提供有效的抗原蛋白。方法 :通过分析BMP - 1类各种分子的基因序列及蛋白结构 ,选择其中的一个片段作为目的基因。从人骨肉瘤细胞株Saos - 2中提取总RNA ,用RT -PCR扩增目的基因片段并克隆到原核表达载体pMALc2中 ,构建成重组质粒BMP - 1(32 2 - 5 88aa) -pMALc2。将该重组质粒转化大肠杆菌DH5 -α ,经过筛选鉴定获得数个阳性克隆 ,再进行DNA序列测定。最后用IPTG诱导宿主菌表达融合蛋白。结果 :BMP - 1基因片段被成功地克隆到pMALc2载体中 ,在IPTG的诱导下能高效表达 ,融合蛋白的表达量占细菌蛋白总量的 (6 6 - 72 ) %。结论 :所获得的重组蛋白包含BMP - 1的几个完整的重要结构域 (2个CUB区和 1个EGF区 ) ,为BMP - 1类分子所共有的高度保守区。该重组蛋白作为抗原 ,可进一步用于产生能有效、特异地识别人类BMP - 1类分子的广谱型BMP - 1抗体。AIM: To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1. METHODS: We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein. RESULTS: The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins. CONCLUSIONS: The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.

关 键 词：骨形态发生蛋白类 基因 融合蛋白 

分 类 号：R363[医药卫生—病理学]