建立人類膜蛋白及分泌蛋白cDNA庫與signal peptide切離位置之理論研究

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研究生：	吳智遠
論文名稱：	建立人類膜蛋白及分泌蛋白cDNA庫與signal peptide切離位置之理論研究
指導教授：	林小喬 Carmay Lim
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2001
畢業學年度：	89
語文別：	英文
中文關鍵詞：	膜蛋白、分泌蛋白、聚合脢鏈反應、切離位置、胺基酸排列順序
外文關鍵詞：	signal peptide, cDNA library, membrane and secreted protein, cleavage site, polymerase chain reaction, PCR, common patterns, residue order
相關次數：	點閱：2 下載：0
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膜蛋白與分泌蛋白（membrane and secreted proteins）在維持生物體正常生理功能上扮演極為重要的角色，參與細胞與細胞間的交互作用、細胞生長與分化、白血球活化與細胞自殺等重要程序，而與免疫調控及疾病密切相關。這兩類蛋白質的胺端（N-terminal）具有一段稱為signal peptide的序列，這段序列主導整個蛋白質分子向內質網（endoplasmic reticulum）運送的過程，在蛋白質分子外送的程序中具有關鍵性的地位，這段序列在膜蛋白與分泌蛋白成熟前會被酵素切斷，其斷裂點稱為切離位置（cleavage site）。由於膜蛋白與分泌蛋白的重要性，取得人類這兩類蛋白的cDNA庫將有助於針對疾病機理及藥物標的的篩選做進一步的研究，而能夠由胺基酸序列準確預測這兩類蛋白質的方法也具有極高的實用性。
我們在現有資料庫中搜尋人類signal peptide的基因序列上長度為9, 12, 15, 18, 21個鹽基的通用片段（common patterns），得到五種特定長度下趨近於最小數目的通用片段組，經由雜訊分析濾去在非signal peptide出現比例太高的片段後，混合得到一組最佳化的解。我們預測利用這一組通用片段做為聚合脢鏈反應（polymerase chain reaction）的啟動子（primer）時，94%以上的啟動子在聚合脢鏈反應中可以得到純度90%以上的膜蛋白與分泌蛋白cDNA產物。另一方面，根據signal peptide切離位置前後胺基酸排列順序的特殊傾向，我們著手發展一套利用此特點預測一蛋白質序列是否具有signal peptide以及其確實切離位置的新方法，在針對1939個分泌蛋白以及1440個不具signal peptide的蛋白質的訓練與測試過程中，初步確定利用切離位置前後胺基酸排列順序的特殊傾向可以精確辨識signal peptide切離位置，其正確率達到94.6%，而判斷一序列是否具有signal peptide的正確率則更高，這個結果顯示胺基酸排列順序的概念具有進一步發展出高準確度signal peptide切離位置或signal peptide預測方法的潛力。

Preparing a human membrane and secreted protein enriched cDNA
  library using a minimal set of PCR primers

1        Introduction

2        Materials and Methods

2.1   Existing databases of cDNAs encoding MSPs and nonMSPs

2.2   Generating l–nucleotide primers

2.3   RT-PCR to clone cDNAs encoding MSPs

2.4   Analyses of primer-derived products

2.5   Estimating the number of primers needed to amplify cDNAs of all human MSPs

3        Results

3.1   The maximal number of l-nucleotide primers needed to amplify all human MSP- cDNAs

3.2   Dependence of the primer set size and noise level on primer length

3.3   Primer composition and features

3.4   Selecting an optimal set of mixed-length primers

3.5   Modifying primers for RT-PCR

4        Discussion

Residue Order-based Prediction of Signal Peptide Cleavage Sites

1    Introduction

2        Materials and Methods

2.1   Residue order-based method

2.2   Criteria for signal peptides and their cleavage site

2.3   Calculations on an existing non-redundant set

3        Results

3.1   Dependence of prediction accuracy on L1 and L2  2.3.2   Accuracy of predicting signal peptide cleavage sites

3.3   Accuracy of identifying signal peptides

4        Discussion

4.1   Dependence of signal peptide cleavage site prediction on the mature protein sequence

4.2   Importance of pairwise residue order

4.3   Future work

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