| Proliferation and differentiation of VECs (human vascular endothelial cells) is crucial to both tissue repair and wound healing after burn and trauma. It is the theoretical and applied basis for acceleration of tissue repair and skin tissue engineering. So it is of great value to study the regulation mechanism of VECs on proliferation and differentiation. Many signal molecules participate in the process. Our previous researches indicated that Inhibitor of differentiation 1 (Id1) could act as a down stream signal of CASK regulating proliferation and differentiation of VECs. Id1 gene codes two different proteins, Id1 protein and Id1' protein. Our previous researches showed that the binding activity of CASK with Id1'is more stronger than that of Id1. Id1 play a major role in negative regulation of cell differentiation and positive regulation of cell growth. It acts as a negative inhibitor of bHLH transcription factors. bHLH factors are divided into two groups: group A, general bHLH proteins, expressed in almost all tissue and cells; group B, tissue-specific bHLH proteins. Id1 can also regulate proliferation and differentiation through non-bHLH pathway. It is not clear whether there are endothelial-specific bHLH /non-bHLH protein or not in VECs. Therefore yeast two hybrid method was employed in this study to screen cDNA libraries with Id1' as bait. To ensure the validity of the results of yeast two hybrid, immunocoprecipitation (IP) was chose to confirm the protein-protein interactions. The main results are as follows:By means of the method of PCR , Id1'cDNA was amplified. Coding sequence (CDS) of Id1'was cloned into bait vector pHybLex/Zeo by recombinant technique. The recombinant bait vector, named as pHybLex/Zeo- Id1',was confirmed correct by sequencing. pHybLex/Zeo-Id1'was transformed into yeast strain EGY48/ pSH18-34 and the transformants were streaked on YC-UZ200 plate and grew well. The result showed that bait fusion protein had no toxic effect on yeast strain. That the transformed strains couldn't grow on YC-ULZ200 media indicated bait fusion protein couldn't nonspecifically activate reporter gene Leu .It was exhibited in β-galactosidase clony-lift filter assay that there was no autonomous activiated reporter gene LacZ in bait fusion protein . The titer of adult human lung cDNA libraries was amplified and determined as2.1(1010cfu/ml. 2 ml of high quality Plasmid DNA was obtained. The purity of cDNA libraries Plasmids is 1.96 and the concentration is measured as 1.1(g/(l.the pHybLex/Zeo-Id1'plasmid and the cDNA library plasmid were sequentially transformed into yeast strain. Transformants were spread on the plates containing YC-WULZ200/Raf-gal media, 198 positive candidate clones expressing Leu+ were obtained. These positive candidate clones were measured byβ-galactosidase clony-lift filter assay and 19 Leu+LacZ+ double positive clones were obtained. Library plasmid DNA was extracted from double positive clones. To exclude false positive clones, all of the double positive library plasmid DNA was transformed into yeast strain respectively with plasmind pHybLex/Zeo-Id1', pHybLex/Zeo-Lamin or pHybLex/Zeo. Finally two true positive clones were obtained.Plasmids of two true positive clones were sequenced .The result indicated that one positive clone was Fyn. Up to now, no paper have indicated that Fyn interacted with Id1'. Another positive clone was Homo sapiens hypothetical protein FLJ10283 and it was named as IDRF. Analyzing the protein strctue of IDRF by informatics, FHA domain and G-patch domain was found. Predicting the function of IDRF, it might play a role in the transduction of DNA damage signals, controlling the cell cycle checkpoint, et al. It was deduced that the interacting of Id1' with IDRF possibly regulate the biological behavior through non-bHLH signals. By means of the methods of RT-PCR, IDRF cDNA was amplified from adult human lung tissue and ECV304 cells. Our results provided evidence for the first time of IDRF expressing in human VECs.To ensure the validity...
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