Detection, cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines
- VernacularTitle:人骨肉瘤细胞株中骨形态发生蛋白-1的检测、克隆和表达
- Author:
Dongying CHEN
;
Quansheng ZHU
;
Chao LIU
;
Jush QIU
- Publication Type:Journal Article
- Keywords:
Bone morphogenetic proteins;
Genes;
Fusion protein
- From:
Chinese Journal of Pathophysiology
1989;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
