人精子甘露糖受体的纯化及其肽质量指纹谱鉴定

发布时间：2018-03-13 13:50

本文选题：人精子　切入点：甘露糖受体　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 受精机理是生命科学的重要理论课题,其阐明对生殖调控具有重大意义,然而受精的分子机制尤其是精卵融合机制,到目前为止并没有完全阐明,人精子甘露糖受体(Mannose receptor,MR)的提出和发现,为人类受精机制的阐明,提供了可能的途径。由于以往的研究仅在于人精子MR的分布、生化特性及生理功能几个方面,为了进一步探讨MR的分子本质,本研究采用甘露糖-琼脂糖凝胶亲和层析法分离纯化人精子MR,纯化蛋白采用自行制备的DMA-Biotin探针进行亲和斑点杂交试验及Western blot以鉴定其甘露糖结合活性。然后,纯化蛋白经胰蛋白酶酶切后,获得的肽段被用于MALDI-TOF-MS分析,利用Mascot搜索引擎对肽质量指纹谱进行数据库检索,其中蛋白质数据库选择SwissProt库。最后,对检索得到的蛋白进行免疫印迹鉴定。结果显示:(1)自行制备的DMA-Biotin探针经亲和斑点杂交试验证明是有效的;(2)纯化蛋白经亲和斑点杂交试验证明具有甘露糖结合活性并且有显著的特异性;(3)纯化蛋白在nonreduce条件下(上样缓冲液不含β-巯基乙醇(2-ME))进行SDS-PAGE,结果可见四条大小分别为27kDa、34kDa、72kDa和121kDa的蛋白条带,在reduce条件下(上样缓冲液含2-ME),可见27kDa、34kda、72kDa蛋白条带,但没有121kDa条带、72kDa蛋白条带染色变浅,而27kDa蛋白条带染色加深。蛋白转膜后用DMA-Biotin探针标记,只有27kDa和72kDa蛋白具有甘露糖结合活性;(4)肽质量指纹谱鉴定27kDa、72kDa和121kDa蛋白均为人血清淀粉样蛋白P成分(SAP)前体,提示72kDa蛋白和121kDa蛋白可能是27kDa蛋白的多聚体;(5)采用抗人SAP抗体进行免疫印迹试验,证实27kDa蛋白为SAP;(6)肽质量指纹谱鉴定,34kDa蛋白为膜联蛋白(ANXA5、ANXA1、ANXA2),但抗人ANXA5抗体的免疫印记试验表明,34kDa蛋白不是ANXA5。综上所述,得出以下结论: 本研究利用甘露糖-琼脂糖凝胶亲和层析方法首次从人精子上分离到了SAP,该蛋白具有甘露糖结合活性,很可能是人精子的甘露糖受体。
[Abstract]:Fertilization mechanism is an important theoretical subject in life science, and its elucidation is of great significance to the regulation of reproduction. However, the molecular mechanism of fertilization, especially the mechanism of sperm-egg fusion, has not been fully clarified so far. The development and discovery of Mannose receptor MRA in human spermatozoa provide a possible way to elucidate the mechanism of human fertilization. Since previous studies only focused on the distribution, biochemical characteristics and physiological functions of human sperm, in order to further explore the molecular nature of Mr, In this study, human sperm MRs were isolated and purified by mannose agarose gel affinity chromatography. The purified protein was used for affinity dot hybridization test and Western blot to identify its mannose binding activity. After the purified protein was digested by trypsin, the peptide fragment was used for MALDI-TOF-MS analysis, and the peptide mass fingerprint was searched by Mascot search engine. The protein database selected SwissProt library. The identified proteins were identified by Western blot. The results showed that the self-made DMA-Biotin probe was proved to be effective by dot-blot hybridization. The purified protein was proved to be of mannose binding activity and had significant specificity in nonreduce. SDS-PAGE was carried out under the condition that the sample buffer did not contain 尾 -mercaptoethanol 2-MEG. The results showed that there were four protein bands with the size of 27kDa 34kDaO72kDa and 121kDa, respectively. Under the condition of reduce, the 27kDa 34kda-72kDa protein band was found in the sample buffer solution, but no 121kDa band was stained shallowly, but the 27kDa protein band staining was deepened. The protein was labeled with DMA-Biotin probe after the protein was transferred to the membrane. Only 27kDa and 72kDa proteins with mannose binding activity were identified as precursors of human serum amyloid P component (SAP) by mass fingerprinting of 27kDa and 121kDa proteins. It is suggested that 72kDa protein and 121kDa protein may be the polymer of 27kDa protein. It was confirmed that 27kDa protein was a SAPXA6) peptide, and the 34-kDa protein was identified as ANXA5, ANXA1, ANXA2, but the immunological imprinting test of anti-human ANXA5 antibody showed that the 34 kDa protein was not ANXA5. On the basis of the foregoing, the following conclusions are drawn:. In this study, SAP was isolated from human sperm by mannose agarose gel affinity chromatography for the first time. This protein has mannose binding activity and may be the mannose receptor of human sperm.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R321.2

【引证文献】