Construction,identification and expression of angiostatin K(1-3) gene eukaryotic expression vector
WU Jing wen1, CHAI Yu bo2,ZHANG Xiang1, GAO Da kuan1,YI Li hnua1, LIU Xian zhen1,WANG Xuan1 1Institute of Neurosurgery of Chinese PLA, Xijing Hospital, 2Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 71003
Aim To construct eukaryotic expression vectorcarrying human angiostatin K(1 3)cDNA and be expressed in cultured gliomas cells. Methods Angiostatin K(1 3) cDNA with secretive signal was cloned into polylinker site of eukaryotic expression vector pcDNA3 to construct pcDNA SAK(1 3),in which the transcription of angiostatin K(1 3) cDNA was driven by CMV promoter; and angiostatin K(1 3) cDNA was identified by restriction enzyme digestion. The vector was transfected into glioma cells,SHG44. The expressed angiostatin K(1 3) protein was identified by electron microscopy and FCM. Its activity was examined by the bovine micrangium endotheliocyte inhibition test. Results The eukaryotic expression vector pcDNA SAK(1 3) was successfully constructed and transfected into glioma cells. The angiostatin K(1 3) protein, which can inhibit endothelial cells proliferation, was obtained. Conclusion The eukaryotic expression vector of functional angiostatin K(1 3) gene is acquired. The vector is of significance for human gliomas-specific gene therapy.