人胚胎干细胞膜的特异多肽受体研究

Study on Receptor of Peptides Specifically Binding Human Embryonic Stem Cell Membrane

目的:通过多肽筛选和比较分析,找到针对人胚胎干细胞(hESC)特异结合的多肽的膜受体蛋白,为相关通路或特异膜表面蛋白下游的研究奠定基础。方法:首先,在前期运用噬菌体展示技术的基础上进行ELISA重筛选,通过对比结合强度的大小,挑选出特异性结合人胚胎干细胞的噬菌体多肽并且进行测序和合成有poly-his标签的多肽;然后运用His Pull-Down系统获得特异结合人胚胎干细胞的某一特殊噬菌体12肽的膜上靶分子受体蛋白;最后质谱测序后通过Mascot数据库和NCBI进行序列信息分析。结果:1通过ELISA重筛选,得到了高特异性结合人胚胎干细胞的两个噬菌体序列,其序列分别为HGAAWGTRTGHV(HGA)和VPATETAQAGHA(VPA)。2通过His Pull-Down实验得到了一个针对多肽VPA的特异性膜蛋白受体。3通过MALD质谱分析以及NCBI的数据库搜索分析,进一步确认这一VPA多肽特异性结合的潜在受体蛋白可能属于HECT超级家族。结论:寻找到一个潜在未知的人胚胎干细胞的特异表面标志物,此膜受体可与VPA多肽特异性结合,为人胚胎干细胞的筛选和鉴定提供了重要指标。

Objective： We used phage screening and analysis to find specific membrane surface receptor on human embryonic stem cells （hESC） which can specifically bind with some unique polypeptides. Then, this study can lead to some further researches on cell signal or pathway. Methods： Firstly, ELISA was used to re-select the special phage peptides which are able to specifically bind hESC by comparing the binding ability between the peptide and hESC, and after sequencing, the poly-his tagged peptides were synthesized. Secondly, His Pull-Down method was used to pull down the target receptor protein which expressed on the hESC membrane and also interacted with our unique 12 peptides. Finally, sequences fi＇om mass spectrometry were fimJaer analyzed with Mascot and NCBI. Results： ① Two special 12 peptides binding specifically with hESC were acquired, and their sequences were HGAAWGTRTGHV （HGA） and VPATETAQAGHA （VPA）. ② We obtained a potential target receptor protein for peptide VPA by His Pull-Down. ③MALD mass spectrometry and NCBI results showed that the supposed target protein might belong to HECT super family. Conclusions： This study found a potential specific marker, for hESC which was able to specially bind with VPA peptide. Most importantly, this work was very useful for screening and identifying hESC.