本文選題：人精子　切入點：甘露糖受體　出處：《福建醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】： 受精機理是生命科學的重要理論課題,其闡明對生殖調(diào)控具有重大意義,然而受精的分子機制尤其是精卵融合機制,到目前為止并沒有完全闡明,人精子甘露糖受體(Mannose receptor,MR)的提出和發(fā)現(xiàn),為人類受精機制的闡明,提供了可能的途徑。 由于以往的研究僅在于人精子MR的分布、生化特性及生理功能幾個方面,為了進一步探討MR的分子本質(zhì),本研究采用甘露糖-瓊脂糖凝膠親和層析法分離純化人精子MR,純化蛋白采用自行制備的DMA-Biotin探針進行親和斑點雜交試驗及Western blot以鑒定其甘露糖結(jié)合活性。然后,純化蛋白經(jīng)胰蛋白酶酶切后,獲得的肽段被用于MALDI-TOF-MS分析,利用Mascot搜索引擎對肽質(zhì)量指紋譜進行數(shù)據(jù)庫檢索,其中蛋白質(zhì)數(shù)據(jù)庫選擇SwissProt庫。最后,對檢索得到的蛋白進行免疫印跡鑒定。 結(jié)果顯示:(1)自行制備的DMA-Biotin探針經(jīng)親和斑點雜交試驗證明是有效的;(2)純化蛋白經(jīng)親和斑點雜交試驗證明具有甘露糖結(jié)合活性并且有顯著的特異性;(3)純化蛋白在nonreduce條件下(上樣緩沖液不含β-巰基乙醇(2-ME))進行SDS-PAGE,結(jié)果可見四條大小分別為27kDa、34kDa、72kDa和121kDa的蛋白條帶,在reduce條件下(上樣緩沖液含2-ME),可見27kDa、34kda、72kDa蛋白條帶,但沒有121kDa條帶、72kDa蛋白條帶染色變淺,而27kDa蛋白條帶染色加深。蛋白轉(zhuǎn)膜后用DMA-Biotin探針標記,只有27kDa和72kDa蛋白具有甘露糖結(jié)合活性;(4)肽質(zhì)量指紋譜鑒定27kDa、72kDa和121kDa蛋白均為人血清淀粉樣蛋白P成分(SAP)前體,提示72kDa蛋白和121kDa蛋白可能是27kDa蛋白的多聚體;(5)采用抗人SAP抗體進行免疫印跡試驗,證實27kDa蛋白為SAP;(6)肽質(zhì)量指紋譜鑒定,34kDa蛋白為膜聯(lián)蛋白(ANXA5、ANXA1、ANXA2),但抗人ANXA5抗體的免疫印記試驗表明,34kDa蛋白不是ANXA5。 綜上所述,得出以下結(jié)論: 本研究利用甘露糖-瓊脂糖凝膠親和層析方法首次從人精子上分離到了SAP,該蛋白具有甘露糖結(jié)合活性,很可能是人精子的甘露糖受體。
[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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R321.2

【引證文獻】

相關碩士學位論文 前2條

1 張艷;人精子甘露糖受體不同探針標記的比較研究[D];福建醫(yī)科大學;2011年

2 李智會;人精子甘露糖受體的分子定性研究[D];福建醫(yī)科大學;2013年

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本文編號：1606650