臺灣博碩士論文加值系統

骨髓細胞上免疫起動受體蛋白 (TREMs) 是由位在人類第六號染色體上與主要組織相容複體 (MHC)相關的基因群表現而來，其中包含活化和抑制型態的受體家族。人類骨髓細胞上免疫受體相似第二型蛋白 (hTLT-2) 為此類蛋白的一種，在先天性和適應性免疫系統中，它是一種會表現在嗜中性白血球、巨噬細胞和B 淋巴細胞上的訊息受體膜蛋白。此蛋白由三百二十一個胺基酸所構成，分子量大約三萬五千道耳吞，然而此蛋白的特性和結構分析並不清楚。在本研究中，我們將hTLT-2蛋白的膜外N端序列分別截取成二萬六千道耳吞 (從第十四號麩胺酸到第二百六十五號天門冬胺酸)和一萬三千道耳吞 (從第十四號麩胺酸到第一百三十二號天門冬�敏i)的部份片段，並選殖到大腸桿菌表現載體pET-28a和pGEX-6P-3，然後表現於不同的大腸桿菌菌種，如BL21 (DE3)、BL21-Gold (DE3)、Rosetta (DE3)。我們在這兩種被截短的hTLT-2部份片段蛋白分別標識His-tag和GST-tag，並以Ni和GSTrap FF 親合性管柱層析 (affinity chromatography) 進行蛋白純化。由實驗結果得知，二萬六千道耳吞膜外截取片段蛋白很難在大腸桿菌系統表現，而且此蛋白容易和包涵體中其它的蛋白聚集。而根據圓二光偏極光譜 (Circular Dichroism spectrum) 的數據，可以得知一萬六千道耳吞帶有His-tag的hTLT-2蛋白片段大約的二級結構是由百分之五的螺旋結構、百分之四十七的β-摺疊結構以及百分之四十八的任意纏繞的結構所組成。所以人類骨髓細胞上免疫受體相似第二型蛋白膜外區域一萬六千道耳吞截取片段的二級結構主要是由β-摺疊結構所組成，與同屬於TREM家族的TREM-1和TLT-1相似。

TREMs, triggering receptor expressed on myeloid cells, belong to a rapidly expanding family of receptors that include activating and inhibitory isoforms encoded by a gene cluster linked to the major histocompatibility complex (MHC) on human chromosome 6. Human TREM-like transcript-2 (hTLT-2), one kind of TREM gene family with 321 amino acids, is a membrane protein and signal receptor expressed on neutrophils, macrophages, and B lymphocytes in innate and adaptive immune system. Until now, the characterization and structural analysis of hTLT-2 remain unclear. In this study, the N-terminus extracellular domain of hTLT-2 was truncated to 26-kDa (from 14-Q to 265-D) and 13-kDa (from 14-Q to 132-N) fragments and cloned into expression vectors pET-28a and pGEX-6P-3. The two partial fragments of hTLT-2 were expressed in competent cells, such as E. coli BL21 (DE3), BL21-Gold (DE3), and Rosetta (DE3). Tag of target proteins were His-tag and GST-tag and the two truncated hTLT-2 proteins were purified with Ni-column and GSTrap FF column, respectively. Guanidine hydrochloride (Gdn-HCl) was used to dissolving the truncated hTLT-2 involved in inclusion body and the target proteins were refolded by dialysis for removing Gdn-HCl in the buffer. The results demonstrate that the larger size of 26-kDa of outer-membrane region of truncated hTLT-2 proteins are difficult to be expressed in E. coli expression system and tend to aggregate with other proteins in inclusion body. According to circular dichroism spectrum, the secondary structure of this 16-kDa of His-tagged hTLT-2 fragment is composed of about 5% alpha helix, 47% beta sheet, and 48% random coil. Thus, the secondary structure of outer-membrane region of 16-kDa of truncated hTLT-2 like TREM-1 and TLT-1 of TREM family mainly comprises beta sheets.

CONTENT

Abstract 1
Abstract (Chinese) 2
Acknowledgement 3
Abbreviations 8
Introduction 10
Research motivation 16
Materials and Methods 17
Materials 17
Cloning of hTLT-2 19
Preparation of plasmids 19
Polymerase Chain Reaction 20
Gel Electrophoresis and Gel Extraction 21
Digestion 22
Ligation 22
Preparation of Competent Cells 22
Transformation 23
Protein Expression 23
Protein Purification 24
His-tagged protein purification 24
GST-tagged protein purification 24
Dialysis and Protein Refolding 25
TCA precipitation of protein 26
SDS-Polyacrylamide Gel Electrophoresis 26
Determination of protein concentration 26
Circular Dichroism spectrometry 27
Result & Discussion 31
Conclusion 49
References 51
Appendix 56

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