Cloning,expression and purification of human canstatin and assay of its bioactivity
- VernacularTitle:人canstatin基因的克隆、表达、纯化及其生物活性测定
- Author:
Xiangrong ZHANG
- Publication Type:Journal Article
- Keywords:
Canstatin;
Clone;
Gene expression;
Protein purification;
Anti-angiogenesis
- From:
Journal of Chongqing Medical University
1986;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To clone human canstatin cDNA,express it as fusion protein in E.coli,purify the fusion protein and determine its biological activity.Methods:Total RNA was extracted from Chinese placenta tissue.The canstatin cDNA was amplified by RT-PCR,then cloned into pTYB1 and sequenced;Prokaryotic expression vector pTYB1-hCAN was constructed;fusion protein was expressed by using IPTG induction and purified by chitin column.The biological activity of the purified fusion protein was identified by using the chick embryo chorioallantoic membrane assay(CAM).Results:RT-PCR product was about 700 bp,its sequence was the same as that of canstatin reported.Prokaryotic expression vector pTYB1-hCAN of human canstatin was constructed successfully,and the fusion canstatin protein was expressed in E.coli.The purity of the protein was 91.6%.The fusion protein could suppress the angiogenesis in CAM.Conclusion:The active human Canstatin is cloned and expressed as a bioactive form.
