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Trial of cloning and functional analysis of a novel vascular endothelial growth factor.

新型血管内皮生长因子的克隆及功能分析试验。

基本信息

批准号：
11671731
负责人：
IWAMOTO Takashi
金额：
$ 2.24万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671731/
关键词：
Interleukin 6 gp130 receptor Tiel TNFα vascular endothelial cell TGFβ 血管内皮

项目摘要

1.Cloning of Interleukin 6(I1-6)receptor/gp130 and TielThe DNA fragment coding the intracellular region of gp130 and that coding the extracellular region of Tiel were amplified by PCR.2.Construction of a chimera gene of gp130 and Tiel and its introduction into pro-B cellsThe DNA fragments of gp130 and Tiel were subcloned into an expression vector of pcDNA3 to construct a chimera gene. Then we transfected this plasmid to IL-3 dependent pro-B cells(Ba/F3)by electroporation method. However, we could not detect the chimera protein by immunoblotting with anit-gp130 antibody. Since RNA expression of a chimera gene was detected, its stability at protein level appeared to be unstable.3.Molecular cloning of the genes that are induced by gp130We cloned several IL-6 induced genes by subtraction method, one of which was Ral guanine nucleotide dissociation stimulator(RalGDS). The induction was fully dependent on Stat3 activation. The analysis of RalGDS function in signal transduction through gp130 now is under way.factor(TNF)α, one vascular endothelial growth factor.4.Cloning of inhibitory molecules for signal transduction of tumor necrosisWhile TNFα induces the apoptosis in NIH3T3 mouse fibroblasts, v-Src-transformed NIH3T3 cells are resistant. We constructed a retroviral cDNA expression library from v-Src-transformed cells and infected it to NIH3T3 cells. Then we selected several clones in the presence of TNFα and recovered cDNA from each clone. One of them was an antisense-fragment coding a novel possible serine threonine phosphatase. Full length of the cDNA was cloned by PCR method, sequenced, and named as PET.When antisense of PET was introduced into NIH3T3 cells, they became partially resistant to TNFα. Now, the analysis of its expression in mouse tissues and function are under way.

1.白细胞介素6（I1-6）受体/gp130和Tiel的克隆利用pcr扩增了编码gp130胞内区和编码Tiel胞外区的DNA片段。gp130和Tiel嵌合体基因的构建及导入原b细胞将gp130和Tiel的DNA片段亚克隆到pcDNA3表达载体中构建嵌合体基因。然后用电穿孔法将该质粒转染到IL-3依赖性的前b细胞（Ba/F3）中。然而，我们用抗gp130抗体免疫印迹法无法检测到嵌合体蛋白。由于检测到嵌合体基因的RNA表达，其在蛋白水平上的稳定性似乎不稳定。gp130诱导基因的分子克隆我们用减法克隆了多个IL-6诱导基因，其中一个是RalGDS （RalGDS）。诱导完全依赖于Stat3的激活。目前正在分析RalGDS在gp130信号转导中的作用。因子(TNF)α，一种血管内皮生长因子。肿瘤坏死信号转导抑制分子的克隆TNFα诱导NIH3T3小鼠成纤维细胞凋亡，v- src转化的NIH3T3细胞具有耐药性。我们从v- src转化细胞中构建逆转录病毒cDNA表达文库，并将其感染NIH3T3细胞。然后，我们选择了几个存在TNFα的克隆，并从每个克隆中提取cDNA。其中一个是一个反义片段，编码一种新的可能的丝氨酸苏氨酸磷酸酶。用PCR方法克隆cDNA全长，测序，命名为PET。将反义PET引入NIH3T3细胞后，NIH3T3细胞对TNFα产生部分耐药。目前，对其在小鼠组织中的表达和功能的分析正在进行中。