The Construction Of Mouse Endostatin's Mutant And The Initial Research Of Its Function

Angiogenesis is the formation of new capillaries, which is the focus ofprogression of solid tumor. The angiogenesis response occurs early intumor development. Under the influence of endogenous angiogenesisinhibitors, metastases remain dormant and tumor cell proliferation isbalanced by an equivalent rate of cell death. And Endostatin has becomewell characterized of the endogenous angiogenesis inhibitors. Endostatin, an endogenous angiogenesis inhabitor, was originallyidentified from the conditioned medium of murine hemangioendotheliomacells by O'Relly et al.. Microsequence analysis of endostatin reveals thatit's a C-terminal cleavage product of collagen ⅩⅧ . The specificfunctional site of endostatin has not been determined yet. There is no reportabout endostatin's analog, but endostatin will retain the native biologicalfunction to inhabit angiogenesis if some amino acids are deleted or added.Some researchs showed that there was no evidence influence onendostatin's function if different polypeptides were added or five aminoacids were deleted from N-terminus of endostatin. But the function has notbeen observed to determine the influence of C-terminal addition or deletionon endostatin biological function in our country. To discuss whether C-terminal sequence affect endostatin biologicalfunction is the point of this research. On the research the primer wasdesigned such that 7 amino acids (FMTSFSK) was deleted from theC-terminal endostatin selectively and got a mutant which we called EM7.And prokaryotic expression vector pBV220-EM7 was constructed by geneengineering. The vectors-pBV220-EM7 and pBV220-endostatin weretransformed into E coli DH5α, purification of the expressed protein wasused to have chick embryo chorioallantoic membrane experiment in vitro.To discuss whether the biological function will be affected by thecomparative study of endostatin and EM7. The main work is as followings: 1.The expressed vector pBV220-endostatin was used as template, andthe EcoRⅠ and BamHⅠ restriction site were introduced into EM7 gene atspecific primer F R, EM7 was amplified by PCR which contained EcoRⅠand BamHⅠ restriction site at its 5'and 3'ends respectively. 2.EcoRⅠ/ BamHⅠ digested PCR products were inserted into thecorresponding restriction site in the expression plasmid pBV220 toconstruct recombinant plasmid pBV220-EM7. Then pBV220-EM7 andpBV220-endostatin were transformed into E.coli. DH5α. The positiveclone were screened and sequenced by the dideoxy chain-terminationmethod. It is consistent with reported sequence. 3.The recombinant mouse endostatin and EM7 genes were expressedin DH5αunder the condition of temperature induction. Then detectexpression with SDS-PAGE. 4.To purify the expressed EM7 and endostatin. And check thebiological function of expressed EM7 and endostatin by CAM. The resultshows that the expressed EM7 and endostatin all can inhibit angiogenesis. The mutant of mouse endostatin-EM7 has been successfully clonedand expression vector pBV220-EM7 has been successfully constructed.pBV220-EM7 and pBV220-endostatin were transformed into E.coli. DH5α. EM7 and endostatin were induced by temperature and purified. TheCAM experiment showed that EM7 can inhibit angiogenesis like endostatin.The research shows that the mutant of mouse endostatin doesn't lost thenative function to inhibit angiogenesis with C-terminal 7 amino aciddeleted. This study is the basis of producting drug for anti-tumor, earlydiagnosis of tumor and endostatin antibody by gene engineering.