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

Angiogenesis is the formation of new capillaries from preexisting capillaries. It is an absolute requirement for the maintenance and progression of overwhelming majority of solid tumors. Exogenous restrictions on angiogenesis severely impair tumor growth and sometimes lead to a complete regression. So the hypothesis that blocking angiogenesis could be a strategy to halt tumor growth came from the pioneering studies of Folkman. The angiogenesis is tightly regulated by a balance of pro-angiogenic factor and anti-angiogenic factor.Endostatin, an endogenous angiogenesis inhabitor, was originally identified from the conditioned medium of murine hemangioendothelioma cells by O'Reilly et al.. Microsequence analysis of endostatin reveals that it's a C-terminal cleavage product of collagen ⅩⅧ which corresponds to the last 184 amino acid residues of the noncollagenous domain.The specific functional site of endostatin has not been determined yet. Some researchers found if 9 and 17 amino acid (NSFMTSFSK, SYIVLCIENSFMTSFSK) were deleted from the COOH terminus of endostatin to form EM1 and EM2 respectively, and the protein of endostatin, EM1 and EM2 were administered to a RCC tumor xenograft model, EM1 protein retained the native biological activity of endostatin , whereas EM2 with an additional 8 amino acids deleted showed loss of function. These suggest that C- terminal conservation may play an important role in endostatin's biological activity. On the research the primer was designed such that 13 amino acids (LCIENSFMTSFSK) was deleted from the C-terminal of endostatin selectively. We have named this mutant EM13. And eukaryotic expression vectors pEGFP-N2-EM13 and pEGFP-N2-Endostatin were constructed by gene engineering. These two vectors were transfected into ascites hepatoma H22 cells and then were used in animal studies. The functions were observed to determine the influence of C-terminal deletion on endostatin biological activity. The main work is as followings:1.Total RNA was extracted from hepatic cells of mouse, Primers were designed and XhoⅠand SacⅡ restriction sites were introduced into them .EM13 gene was cloned by RT-PCR and contained XhoⅠand SacⅡ restriction sites at its 5' and 3' ends respectively.2.The PCR product was linked with pMD18-T vector and was transformed into E.coli DH5α.The positive clone was screened and sequenced by the dideoxy chain – termination method. It is consistent with reported sequence.3.The pMD18-T-EM13 was digested by restriction enzyme XhoⅠand SacⅡ and then was inserted into the corresponding restriction site in the espression vector pEGFP-N2. Eukaryotic expression vector pEGFP-N2-EM13 was constructed. And pEGFP-N2-Endostatin was constructed too.4.The recombinant plasmids carrying EM13 and endostatin were transfected in ascites hepatoma H22 cells using the lipofectAMINE method. The transfected and selected cells were implanted subcutaneously into Balb/c mice respectively. The results indicated that compared with non-transfected group ,the group inoculated with H22 cells which transfected with endostatin proved no significant difference but developed relatively small tumor and relatively low density of blood vessel, which suggests endostatin gene may decrease the tumorgenesis of H22 cells. Mice inoculated with H22 cells which transfected with EM13 have no difference with the mice of non-transfected group, which suggests that EM13 gene has no effect on the tumorgenesis of H22 cells.The mutant of mouse endostatin—EM13 has been successfully cloned and expression vectors pEGFP-N2-EM13, pEGFP-N2-Endostatin has been successfully constructed. These two vectors were transfected into ascites hepatoma H22 cells and then were used in animal studies. The results suggested the EM13 gene may lose of native function because of the deletion of C-terminal amino acids, the several amino acids of COOH terminus may play an important role in endostatin's biological activity. Further researches on its specific effects and mechanisms are needed.