臺灣博碩士論文加值系統

Interleukin-8 (IL8), a kind of CXC chemokines with an ELR motif, is a
novel type of neutrophil-activating cytokine. IL8 with small molecular weight of
8.5 kDa plays a pivotal role in inflammatory response. Overexpression of IL8
would cause acute inflammatory response. Thus it may be a therapeutic target to
the antagonist. Professor John Gordon indicates that bovine IL8(3-74) K11R/G31P
mutation is a potential antagonist and the humanized bovine IL8(3-74)
K11R/G31P(hbG31P) also remain the ability of antagonist . .However, the
structure of it was still unknown. In this study , we investigated structural of
hbG31P. The backbone sequential assignments were decide with five triple
resonance experiments (HNCA, HNCO, HNCACO, CBCANH, and
CBCA(CO)NH). Side chain assignments were also determined with
HBHACONH, HCC(CO)NH and HCCH-TOCSY. Secondary structures were
predicted via PSICSI analyses. These results were compared with human
K11R/G31P and indicated that the pattern of secondary structure is similar to
human IL8 K11R/G31P. However, their function were opposing to wildtype IL-8.
We suggest that the cis-trans conformation of Pro31 and Pro play an important
roller. Furthermore, we work toward the purpose.

介白素-8（Interleukin-8, IL8）為一72 個胺基酸組成，分
子量約為8.5 千道耳吞的蛋白質。由於IL8 具有引起白血球化學傾向
的能力，能誘導發炎反應，因此其和許多發炎性疾病有關,因此過多
的介白素反應會造成一些過度發炎反應。John Gordon 教授在2002
年發現，對小牛IL8 進行K11R/G31P 兩個點突變後，此突變的小牛
IL8 相似物對IL8 具有功能上的拮抗作用，之後也發現人類化的小牛
IL8 也具有此拮抗作用。因此我們決定利用核磁共振儀，對此相似物
進行結構上的分析。利用HNCA、HNCO、HNCACO、CBCANH 和
CBCA(CO)NH 光譜，我們可以得到此相似物之骨架序列光譜的前後
標定順序關係，而胺基酸上側鏈的氫訊號則是經由HBHACONH、
HCCH-TCOSY 以及HCC(CO)NH 等光譜實驗來觀測。除此之外，
我們利用上述得到的結果做化學位移索引（CSI）之統合分析，然後
跟人類的的介白素相似物做比較，發現他們二級結構組成相似。但是
它們卻擁有跟介白素相反的功能，所以我們推論或許是因為在脯胺酸
31 和脯胺酸32 正反向異購物的組成有很大的關係。目前我們也朝這
個目標進行實驗。

ABSTRACT ...........................................................................................................................................1
摘要.........................................................................................................................................................2
CHAPTER 1 INTRODUCTION........................................................................................................3
CYTOKINE............................................................................................................................................3
CHEMOKINES ......................................................................................................................................3
INTERLEUKIN-8(IL8/CXCL8)..............................................................................................................4
ANTAGONIST .......................................................................................................................................5
CHAPTER 2 MATERIALS AND METHODS .................................................................................7
PREPARATION AND PURIFICATION OF PROTEIN SAMPLE....................................................................7
A. Construction of K11R/G31P Expression Plasmid ....................................................................7
B. Preparation of competent cell ...................................................................................................8
C. Transformation.........................................................................................................................8
D. Expression of hbG31P...............................................................................................................9
E. Isotope labeling of sample for NMR.......................................................................................10
F. Purification of hbG31P............................................................................................................10
CIRCULAR DICHROISM SPECTROSCOPY (CD) ..................................................................................... 11
PREDICTION OF PERCENTAGES OF PROTEIN SECONDARY STRUCTURE FROM CD SPECTRA...........13
MASS SPECTROMETRY.....................................................................................................................13
NMR SPECTROSCOPY ........................................................................................................................14
NMR EXPERIMENTS ..........................................................................................................................15
DATA PROCESSING AND ANALYSIS......................................................................................................15
THE SECONDARY STRUCTURE PREDICTION OF CHEMICAL SHIFT INDEX.........................................16
CHAPTER 3 RESULTS.......................................................................................................................17
EXPRESSION, PURIFICATION AND PROPERTIES OF PROTEIN SAMPLE .................................................17
AMINO ACID SEQUENCE ALIGNMENT OF K11R/G31P.......................................................................18
CONFORMATIONAL ANALYSIS BASED ON CIRCULAR DICHROISM (CD) OF HBG31P.....................18
RESONANCE ASSIGNMENT ................................................................................................................19
Backbone assignments .................................................................................................................19
Side chain assignments ................................................................................................................20
PREDICTION OF CHEMICAL SHIFT INDEX (CSI) ...............................................................................21
NOE ASSIGNMENT............................................................................................................................22
CHAPTER4 DISCUSSION..............................................................................................................23
FIGURES ...........................................................................................................................................25
TABLES ...............................................................................................................................................43
REFERENCE ......................................................................................................................................47

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