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Cloning and Expression of Human Canstatin and Identification of Its Biological Activity
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Abstract:
Objective: To clone and to express human canstatin gene and investigate biological activity of the recombinant protein.Methods: The cDNA of canstatin was amplified with RT-PCR from fresh fetal liver and was cloned into pMD18-T vector , and was sequenced.Then canstatin cDNA was cloned into the BamHⅠand HindⅢ sites of pQE30 and expressed with induction of IPTG. After the purification under native conditions, The biological activity of the recombinant protein was identified by the chick embryo chorioallantoic membrane assay (CAM). Results:The sequence of the amplified DNA fragment is consistent with that of the known gene. The recombinant protein was highly expressed after induction with IPTG. Biological assay results indicate that the recombinant canstatin protein could suppress the new blood vessel formation in CAM in vitro.Conclusion: The cloning and expression vector of canstatin cDNA has been constructed successfully. The recombinant canstatin protein was highly expressed in E.coli M15. In the CAM, the recombinant protein has highly suppressive effects on the vessels in chick embryo chorioallantoic membrane.
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