臺灣博碩士論文加值系統

雞的組織相容性複體（又稱為B-complex）的功能是負責抗原的辨識與呈現，此乃免疫系統啟動之關鍵。過去有關B-G抗原的研究以來亨蛋雞（少數以肉雞）為主要研究對象，其功能仍不十分明白，但一般常被用來當作雞的B-complex分類之依據。本研究目的是藉由B-G抗extracellular domain序列分析，定出台灣土雞近親品系B-G抗原之haplotypes。以Miller所發表B-G基因extracellular domain之序列為藍本，取其保留區域設計出特異性引子對（gene specific primers:BG-1、BG-2），以增幅台灣土雞(近親品系7、9、11、12，每品系雌雄各5隻)的genomic DNA，結果得到符合預期的330 bp DNA片段。將此片段作T-A cloning，並隨機選取7~10個clones，萃取plasmid後作DNA自動定序分析；所得之序列資料與GenBank中之資料（gb: M61864）比對，結果相似度高達95％，顯示選殖之DNA片段即為B-G基因extracellular domain。又隨機選取一土雞（T-13649）盡可能的挑選clones並作DNA定序，發現有8種haplotypes，顯示台灣土雞B-G基因至少有8個loci。將四種品系之台灣土雞以分析軟體DAMBE（Data Analysis in Molecular Biology and Evolution）作分析，發現經過人工選拔之台灣土雞，其B-G基因可分為6群組（G1~G6）。繼而統計分析此6群組與性別、品系間的關聯性：B-G抗原的胺基酸haplotypes 在性別上沒有顯著差異（P＞0.05），品系間亦無顯著差異（P＞0.05）；但是群組間有顯著差異（P＜0.05），品系與群組間有交感效應之趨勢（P=0.05）。

The major histocompatibility complex, also known as B-complex in the chicken, has a major influence no controlling disease resistance and immune responses. In the past, many of the B-G antigens (chicken MHC class Ⅳ) in Leghorn lines of chicken had been studied. Although the functions of B-G antigens are not well known, B-G antigens are served as bases to classify B-complex. The objective of this study is to define B-G antigen genotypes in Taiwan native chicken by analyzing the amino acid sequences of B-G antigen extracellular domain. PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding extracellular domain of B-G antigen. By using genomic DNA of Taiwan native chicken of（line 7, 9, 11, 12）with PCR, the DNA fragments（~330 bp）were obtained as expectrd. After T-A cloning, plasmid purification, DNA sequencing and aligning with GenBank, it was found that the similarity between DNA fragment and B-G mRNA sequence （gb: M61864）is 95%, implying that the DNA fragment is extracellular domain of B-G antigen. In addition, 8 B-G antigen were discovered haplotypes after random sampling Taiwan native chicken (T-13649), by picking the clones (from T-A cloning) as many as possible, and sequencing DNA as well. It indicated that B-G antigen haplotypes are no less than 8 loci of individual chicken. We defined 6 groups（G1~G6）of B-G antigen haplotypes with analyzing the amino acid sequences in 4 lines of Taiwan native chicken. Analysis of B-G antigen haplotypes between sex, lines and groups. The results showed there is no significant difference between sex and lines (＞0.05); B-G antigen haplotypes are significantly different in groups (P＜0.05); and there is a correlation between lines and groups（P=0.05）.

目 錄
頁數
中文摘要 ………………………………………………………… Ⅰ
英文摘要 ………………………………………………………… Ⅲ
英文縮寫表 ……………………………………………………… Ⅴ
致謝 ……………………………………………………………… Ⅵ
目錄 ……………………………………………………………… Ⅶ
圖次索引 ………………………………………………………… Ⅸ
表索引 …………………………………………………………… Ⅹ
附錄索引 ………………………………………………………… XI
第一章 前言……………………………………………………… 1
第二章 文獻探討………………………………………………… 3
2.1 哺乳動物之組織相容性複體 ……………………………… 3
2.2 家禽之組織相容性複體 …………………………………… 5
2.2.1 B-F組織相容性複體（chicken MHC class I ）……… 5
2.2.2 B-L組織相容性複體（chicken MHC class Ⅱ）……… 6
2.2.3 B-G組織相容性複體（chicken MHC class Ⅳ）……… 6
2.3 家禽組織相容性複體之檢定 ……………………………… 12
2.4 家禽組織相容性複體之抗病性 …………………………… 13
第三章 材料與方法 …………………………………………… 16
3.1 試驗動物 …………………………………………………… 16
3.2 血液樣本收集與染色體DNA萃取…………………………… 16
3.2.1 血液樣本收集 …………………………………………… 16
3.2.2 染色體DNA萃取…………………………………………… 16
3.3 引子設計與聚合酶鏈鎖反應 ……………………………… 17
3.3.1 引子設 …………………………………………………… 17
3.3.2 Gradient PCR …………………………………………… 18
3.3.3 PCR產物純化……………………………………………… 18
3.4 TA cloning與大腸桿菌轉殖作用 ………………………… 19
3.4.1 接合反應 ………………………………………………… 19
3.4.2 轉殖實驗 ………………………………………………… 20
3.5 質體DNA萃取………………………………………………… 20
3.6 序列分析 …………………………………………………… 21
3.6.1 Minimum Evolution（ME）method……………………… 22
3.6.2 Neighbor-Joining (NJ) Method ……………………… 22
3.6.3 Maximum Parsimony (MP) Method……………………… 23
3.7 統計分析 …………………………………………………… 23
第四章 實驗結果 ………………………………………………… 24
4.1 Gradient PCR………………………………………………… 24
4.2 PCR Screening ……………………………………………… 24
4.3 DNA及胺基酸序列比對 ……………………………………… 24
4.4多重序列並列分析 …………………………………………… 28
4.5親源關係樹繪製與序列分組 ………………………………… 28
4.6統計分析 ……………………………………………………… 42
第五章 討論 ……………………………………………………… 46
參考文獻…………………………………………………………… 50
作者簡介…………………………………………………………… 59
附錄………………………………………………………………… 60

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