Construction Of Human Canstatin Gene Secretory Eukaryotic Expression Vector And Identifing Anti-angiogenesis Biological Activity Of The Expression Products

[Objectives] To construct human canstatin gene secretory eukaryotic expression vector. To identify anti-angiogenesis biological activity of the supernatant after transfecting CHO-Kl cells.[Methods] The cDNA of human canstatin was amplified with RT-PCR from human placent umbilical cord and was inserted into cloning vector pUCm-T with T-A cloning. After transformation into E.coli DH5a, the recombinant plasmid was analyzed by restriction endonucleases and sequencing to confirm the obtained cDNA fragment was consistent with published sequence. Canstatin cDNA fragment was subcloned from pUCm-T vector into the BamH I and HindIII sites of pSecTag2B secretory eukaryotic expression vector. 72h after transient transfecting CHO-K1 cells, RT-PCR assay was performed to detect the expression of canstatin gene in CHO-Kl cells. The proteins in cells and in supernatant were collected respectively and were analyzed by SDS-PAGE electrophoresis. The anti-angiogenesis biological activity of the supernatant protein was identified by chick embryo chorioallantoic membrane (CAM) assay.[Results] The 684bp cDNA fragment of canstatin was obtained successfully . The cloned cDNA coding sequence was shown to be consistent with reported sequence derived from Genbank. Secretory eukaryotic expression vector of recombinant human canstatin gene was successfully constructed and was used to transient transfect CHO-Kl cells using positive ion liposome method. RT-PCR assay showed that the canstatin containing plasmid was successfully transfected into CHO-Kl cells. After centrifuging and collecting the proteins in cells and in supernatant, there were both a clear protein band at about 30kD site in SDS-PAGE, which was similar to the...