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研究生:陳詩欣
研究生(外文):Hsih-Hsin Chen
論文名稱:膜鍵結免疫球蛋白E的CεmX區段於靈長類物種的遺傳序列分析
論文名稱(外文):Genetic analysis of CεmX segment in mIgE in human and non-human primate species
指導教授:張子文張子文引用關係
指導教授(外文):Tse-Wen Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:46
中文關鍵詞:膜鍵結免疫球蛋白EC(epsilon)mX 區段靈長類遺傳同源性
外文關鍵詞:membrane-bound IgEC(epsilon)mXnon-human primateshomology
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免疫球蛋白E (IgE) 是引起第一型過敏性免疫反應的主要媒介。當肥大細胞及嗜鹼性球細胞表面的高親和性受器 (Fc-epsilon RI) 所結合的IgE與過敏原 (allergen) 辨識,促成IgE的交聯 (cross-linking),以及 Fc-epsilon RI 的聚集 (aggregation),瞬時激發這些細胞釋放組織胺 (histamine)、白細胞素 (leukotrienes) 等藥理媒介物質(pharmacological mediators),造成多種的發炎反應。
本論文重點在於探討膜鍵結免疫球蛋白E (mIgE) 的 C(epsilon)mX 區段於靈長類物種的遺傳分析;此項研究結果將能有助於了解膜鍵結免疫球蛋白E的 C(epsilon)mX 區段於靈長類物種的演化關係。C(epsilon)mX 區段位於mIgE 其重鏈固定區域CH4結構區及穿膜區胜肽鏈之間,由52個氨基酸組成。目前只在人類及其他靈長類找到,但其他較低等的動物則不存在。
在此特別針對非人靈長類進行更深入的有關 C(epsilon)mX 基因型研究,物種包括黑猩猩、大猩猩、紅毛猩猩、長臂猿、東非狒狒跟台灣獼猴。由全血樣本中分離出週邊血液單核球(PBMC)經提取RNA與DNA用於PCR,擴增了mIgE上 C(epsilon)mX 結構域的DNA片段,獲得的PCR産物則進行DNA序列分析。各物種 C(epsilon)mX 區段核甘酸與正常人比對後發現相似度分別為 99.75 %、100 %、95.02 %、94.03 %、90.88 % 及 89.88 %。由NCBI blastx (http: //www.ncbi.nlm.nih.gov/BLAST/),一級氨基酸序列相似度為98.51 %、100 %、93.28 %、88.06 %、85.07 % 及 85.07 %。又可於台灣獼猴的 C(epsilon)mX 區段第21、 31、 33、 36、 37、 38、 51、 63、 65與66等位置發現性質差異度較大的氨基酸取代情形,這些變異的生理意義目前並不清楚。
本研究歸納出 C(epsilon)mX 區段存在於人之外其他六種非人類靈長類,DNA 與蛋白質序列中均有高度的同源性 (homology),能提供 C(epsilon)mX 分子演化研究的基礎。再者對台灣特有種─台灣獼猴 (Formosan macaque) 進行較大族群的序列分析,期能成為日後 C(epsilon)mX 研究上適當的動物模式。
C(epsilon)mX 三級結構的解出;針對 C(epsilon)mX 區段核甘酸序列於不同族群的基因多型性研究將有助於了解過敏性免疫疾病引發歷程中,C(epsilon)mX 可能扮演的角色,尤其是它對表現mIgE的B細胞及IgE的產生的調控於發展疾病治療機制過程中, C(epsilon)mX 存在的獨特性提供了應用潛力很大的免疫藥物標定目標。
Immunoglobulin E (IgE) is a central mediator of immediate-type hypersensitivity reactions. Our laboratory previously discovered that the membrane-bound IgE (mIgE) on human B cells contains an extra peptide segment of 52 amino acid residues, referred to as C(epsilon)mX, between the CH4 domain and the membrane-anchor peptide. The sequence of C(epsilon)mX is unique in the entire protein bank. While the function and possible roles of C(epsilon)mX in the regulation of IgE production remain unknown, C(epsilon)mX has provided as a potential site for immunological targeting of IgE-expressing B cells for the treatment of allergic diseases.
In the present study, we analyze the C(epsilon)mX in human and several non-human primates, as a way to understand the function of C(epsilon)mX. We have cloned the C(epsilon)mX segment in genomic DNA (gDNA) and cDNA from chimpanzee, gorilla, orangutan, gibbon, olive baboon and Formosan macaque, using PCR and primers designed from the sequences of human C(epsilon)mX and its neighboring segments. Between human and the above listed six non-human primate species, the C(epsilon)mX share homology of 98.51 %, 100 %, 93.28 %, 88.06 %, 85.07 % and 85.07 % in amino acid sequences, respectively. Sequencing data showed that in addition to some variations within the C(epsilon)mX segment, there are also variations in the adjacent extracellular segment, referred as migis-epsilon, of the membrane-anchor peptide. These variations include Gly21Glu, Gly31Arg, Ser33Pro, His36Asp, Pro37His, Arg38His, His40Arg, Pro51Leu, Val56Leu, Gly63Ser, Ala65Val and Pro66Leu (between human and Formosan macaque).
We are in progress studying 20 individuals of Formosan macaque to analyze potential allotypic variations in C(epsilon)mX and migis-epsilon. The information on the genetic variation among human and non-human species may help us understand the function and structure of those segments and their roles in the regulation of IgE. The results may also be used to evaluate the suitability of using certain non-human primate species, such as Formosan macaque, as an animal model for studying the therapeutic targeting of IgE-expressing B cells using immunological agents that aim at C(epsilon)mX.
Introduction ……………………………………………………………8

Materials and Methods
Preparation of blood samples………………………………………11
Extraction of RNA and reverse transcription (RT)……………11
Extraction of genomic DNA …………………………………………12
PCR amplification ……………………………………………………12
Cloning of a DNA segment of C(epsilon)mX………………………13
Data analyses …………………………………………………………14

Results
Alignment of C(epsilon)mX sequence………………………………15
C(epsilon)mX sequence derived from non-human primates ……16
Sequence divergence of C(epsilon)mX on C epsilon 1�����n gene and C(epsilon)mX domain………………………………………17
Phylogenetic analysis of C(epsilon)mX of C�惀psilon 1�nGene………………………………………………………………………17

Discussion………………………………………………………………18

Tables
Table 1 Blood sample…………………………………………………21
Table 2 PCR primer-pairs……………………………………………22
Table 3 Sequence variations of human genomic C(epsilon)mX segment …………………………………………………………………23
Table 4 Sequence variations of non-human primate genomic C(epsilon)mX segment …………………………………………………28
Table 5 Sequence variations of (epsilon)m67 peptide ………33
Table 6 A cDNA sequence comparison between the upstream and downstream sequences of target segment and human C�惀psilon 1 gene sequence ………………………………………………………36
Table 7 A gDNA sequence comparison between the upstream and downstream sequences of target segment and human C�惀psilon 1 gene sequence ………………………………………………………37
Table 8 The epitope recognized by A20 …………………………38

Figures
Figure 1 The location and prototype sequence of C(epsilon)mX in human mIgE………………………………………………………39
Figure 2 Multiple mRNA encoding for different human epsilon isoforms…………………………………………………………………40
Figure 3 The phylogenetic relationship…………………………41
Figure 4 A proposed model of disulfide bonds in C(epsilon)mX amd migis-epsilon regions………………………………………42

References………………………………………………………………43
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