臺灣博碩士論文加值系統

中文摘要

蛋白質晶片時常都被用來偵測我們所感興趣的病原分子。但蛋白質晶片都會面臨到抗體方向性不固定以及抗體彼此空間分布不均導致空間障礙使得檢測效率降低。本篇論文研究想利用自組裝模方式結合C端帶有組胺酸的蛋白A的方式來解決以上蛋白質晶片所面臨到的問題。
我們選用了長短鍊混合硫醇類自組裝的方式希望達到空間分布均勻的效果。長鏈硫醇類選用11個碳化學官能基為COOH來結合蛋白質，而短鏈硫醇類選用5個碳化學官能基為CH3避免蛋白質結合。之前學長研究指出在50%混和比例當中分布比例最佳。我們在蛋白A 的尾端修飾組胺酸用來固定蛋白A方向性，且藉由蛋白A只接抗體Fc端的特性來固定抗體方向性。本實驗想同時藉由混和硫醇類以及蛋白A尾端修飾組胺酸來同時克服抗體方向性以及抗體空間障礙的問題以提高蛋白質晶片的檢測效率。
在本篇報導中，50%混合硫醇類那組蛋白A的結合抗體效率為7.9%而尾端有修飾組胺酸的蛋白A結合抗體效率為18.7%，在100%混合硫醇類蛋白A結合效率為29.9%而尾端有修飾組胺酸的蛋白A抗體結合效率為31.6%。可知有固定方向性的蛋白A的結合效率較好，但在表面修飾分布比例來取開蛋白兼具以提高抗體結合效率這部分，卻不如我們預期，此部分將是我們未來要改善的目標。

Table of content
Abstract in Chinese…………………………………………….1

Abstract…………………………………………………...........3

Abbreviations…………………………………………………..7

Introduction………………………………………………….....9

Materials and Methods………………………………………..15

Results…………………………………………………………23

Discussion……………………………………………………..29

References……………………………………………………..33



Figures and Tables………………………………………….. 35
Figure 1. DNA manipulation……………………………….....36
Figure 2. Fluorescense intensity on microarray via the protein modified by mixed thiol group………………………………..37
Figure 3. Fluorescence intensity on microarray via recombinant
protein A modified by Ni-NTA group………………………....38
Figure 4. Molecular cloning of spa……………………………39
Figure 5. Construction of pET23a…………………………….40
Figure 6. Ligation of pET23a and spa……………………………41
Figure 7. Different induction time for overexpression of
recombinant protein A…………………………………...........42
Figure 8. Assay activity for IgG binding……………………...43
Figure 9. Purification of recombinant protein A……………....44
Figure 10. Purification of recombinant protein A……………..45
Figure 11. Binding of IgG on protein chip by protein A as
determined by fluorescence…………………………………...46
Figure 12. Intensity of fluorescence on microarray…………...47
Figure 13. Intensity of fluorescence on microarray…………...48

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