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研究生：	林修弘 Lin, Hsiu-Hung
論文名稱：	人類HBsAg 基因載體之構築及轉染雞細胞之研究 Construction of HBsAg Plasmid and Transfection of Chicken Primordial Germ Cells and Spermatozoa
指導教授：	戴謙 Tai, Chein
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	70
中文關鍵詞：	家禽、電穿孔法、B 型肝炎、始基生殖細胞、食物疫苗
外文關鍵詞：	primordial germ cells, PGCs, HBsAg, edible vaccines, chicken, electroporation
相關次數：	點閱：104 下載：1
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食物疫苗因為有其方便利用之特性而受到研究上的重視，而B型肝炎為中國人的一項重要傳染病，在台灣之臨床經驗及研究相當進步，正確檢驗方法亦建立已久，因此利用食物疫苗預防B型肝炎之可行性值得探討。本論文之目的即在建立利用家禽作為人類B型肝炎病毒表面抗原(HBsAg) 生物工廠的試驗方法。
試驗中將HBsAg基因構築於含CMV 啟動子之載體 pEGFP–N1中，並將所建構之 pEGFP-N1-HBsAg 載體轉殖入白色來亨雞性腺細胞，分離其中之始基生殖細胞（primordial germ cell, PGC），並注入土雞stage 13-15之胚胎背大動脈中，以得到具有轉殖基因之生殖系嵌合體的雞隻，育成後將可透過後裔毛色測定，選出帶有此一外源性基因之後裔。另將建構之 pEGFP-N1-HBsAg 載體轉殖入雞的精子中，再對母雞進行人工授精，以期得到帶有外源HBsAg 基因的雞隻。使用兩種方法進行，希望能得到表現外源基因的轉殖個體，並可利用此轉殖個體生產帶有外源蛋白的雞蛋。
在始基生殖細胞的轉殖結果發現性腺細胞中及細胞培養液中皆有HBsAg基因的存在，證實HBsAg 基因可在性腺細胞中表現。進而將轉殖後的始基生殖細胞注射入雞胚中，發現stage 26-28 的胚胎性腺亦有HBsAg 基因之存在。於 68 個雞胚胎性腺中共檢測出有 20 個樣本含有 HBsAg 基因，證實經電穿孔後的 PGC 依然保有移行到性腺的功能，而轉殖成功率約為 29.4%。
雞精子於轉殖pEGFP-N1-HBsAg後進行人工授精，並於雛雞孵出兩星期後採血萃取 DNA，PCR 檢測結果38隻雛雞中有 25 隻可檢測出HBsAg 基因，HBsAg 基因轉殖成功率為60.5%。
不論經由始基生殖細胞移轉胚胎性腺或轉殖精子所生雛雞之DNA，經PCR反應所得之複製產物，在定序分析後與HBsAg 基因相似度為96-98%，表示HBsAg 基因確實已轉入雞性腺細胞及透過精子傳至雛雞中。雛雞基因表現經ELISA分析，初步在其血清中發現有HBsAg 陽性反應，但對照血清亦出現交叉反應，顯示有待更進一步確認。
本試驗結果顯示以始基生殖細胞或雞精子轉殖pEGFP-N1-HBsAg皆可轉入HBsAg 基因，若轉殖基因個體確定可以穩定的表現外源蛋白至雞蛋中，則以每隻雞每年1kg的雞蛋蛋白質產量，應可利用此方法供應大量的藥用蛋白，或直接以雞蛋作為食物疫苗，提供非侵入性的疫苗使用方法。

Edible vaccines have been studied in transgenic plants, and it has been shown that transgenic plant containing human hepatitis B virus antigen (HBsAg) was able to induce a primary immune response. This study is going to establish the protocols for producing HBsAg by poultry egg.
The HBsAg gene was construced into the vector of pEGFP-N1 and transfected into the cells of gonads of chicken embryos at stage 26-28 by electroporation. After three days of culture, the cells pellet which contained primordial germ cells (PGCs) and the cultured medium of transfected cells were analysed for the production of HBsAg by ELISA. HBsAg was detected both in cell pellet and culture medium. In addition, freshly transfected PGCs were also transferred to the recipient embryos, by microinjection through the dorsal aorta of embryos at stage 13 to stage 16, to obtain the germ-line chimera. The gonadal ridges of day-7 embryos were analysed for the HBsAg gene by PCR and DNA sequencing. Twenty out of 68 gonad samples showed positive result in PCR analysis, and the transfection rate was 29.4% .The identities to HBsAg gene is 96%. Further studies will be needed to examine the germ-line chimerism of PGC transferred chicken by the progeny plumage test, and to detect the possibility of gene transfer if it is a donor-derived progeny from a germ-line chimera.
The pEGFP-N1-HBsAg plasmid was also transfected into the spermatozoa of chicken by electroporation. DNA of chicks obtained from the artificial insemination of transfected spermatozoa was analysed for the integration of HBsAg gene by PCR and DNA sequencing, and the serum was analysed for the production of HBsAg by ELISA. Among 38 chicks, 25 showed positive PCR reaction, indicating a 60.5% transfection rate. Although some serum samples of chicks exhibited positive reaction on ELISA analyses, false-positive results were also found in control sera. The gene expression in the serum of gene transfer chicken remained to be clarified.
The PCR analysis and DNA sequencing confirmed the positive integration of HBsAg gene in the gonads of recipient embryos after transfer of primordial gem cells, and it was also found in the 2-week chicks of first generation derived from sperm-mediated gene transfer. These results provide a potential production route of edible vaccine of HBsAg through the gene transfer in chicken.

摘要............................................I
英文摘要......................................III
致謝............................................V
目錄...........................................VI
附錄...........................................IX
圖目錄..........................................X
表目錄.........................................XI
縮寫對照表....................................XII
前言............................................1
文獻檢討........................................3
1. B 型肝炎病毒概述.............................3
1.1. B 型肝炎病毒 (HBV).........................3
1.2. B 型肝炎表面抗原
(Hepatitis B surface antigen)...................5
1.2.1 Small Hepatitis B surface antigen 
( HBsAgorSHBsAg) ...............................8
1.2.2 Middle Hepatitis B surface antigen 
( MHBsAg )......................................8
1.2.3 Large Hepatitis B surface antigen 
( LHBsAg ).....................................10
1.3. B 型肝炎疫苗..............................10
2. 家禽基因轉殖方法............................11
2.1. 反轉錄病毒轉殖法 
(Retrovirus transfection)......................11
2.2. 脂轉殖法 (lipofection)....................12
2.3. 電穿孔轉殖法 (electroporation)............13
2.3.1. 電穿孔轉殖PGC...........................15
2.3.2. 電穿孔轉殖精子..........................16

3. 食物疫苗（edible vaccine）概述..............17
3.1 植物食物疫苗...............................18
3.2 動物食物疫苗的研究.........................19
材料方法.......................................20
1.試驗材料.....................................20
1.1性腺始基生殖細胞提供胚胎及接受胚胎，
及提供精子及接受精子的品種.....................20
1.2 載體.......................................20
1.3 大量純化載體的套組.........................20
1.4 電穿孔系統.................................20
1.5 酵素連結免疫分析法(ELISA)套組..............21
1.6 瓊酯膠 DNA 片段純化套組....................21
1.7 DNA 萃取套組...............................21
2. 大量製備 pEGFP-N1 載體......................21
2.1大量純化pEGFP-N1............................22
3.構築 pEGFP-N1-HBsAg 載體.....................22
3.1擴增 HBsAg 基因片段.........................22
3.2自瓊酯膠體中純化 DNA 片段...................24
3.3以限制脢剪切DNA 片段與載體(Cutting).........25
3.4 DNA 片段與載體的接合作用(Ligation).........25
3.5 將 pEGFP-N1-HBsAg 轉殖入  
competent cell 中..............................27
4.電穿孔轉殖始基生殖細胞.......................27
4.1 收集性腺(無菌操作).........................27
4.2 分離性腺細胞...............................28
4.3 電穿孔轉殖性腺細胞.........................30
5.以 ELISA 套組測定 HBsAg 的表現...............31
5.1 沈澱培養液中的蛋白質.......................31
5.2 ELISA 套組測定 HBsAg.......................31
6. 顯微注入stage14-16 土雞胚胎.................32
6.1 分離來亨雞胚胎血液中的cPGC.................32
6.2 顯微注入stage14-16 土雞胚胎................33
7. 電穿孔轉殖精子..............................33
8. 基因序列比對................................33
結果與討論.....................................34
1. 構築 pEGFP-N1-HBsAg 載體....................34
2. 測定pEGFP-N1-HBsAg 載體在性腺細胞中的表現...38
3. 檢測轉殖 PGC 胚胎的性腺.....................39
4. 檢測介由精子轉殖所生後代之基因表現..........45
4.1 以PCR 檢測 DNA.............................45
4.2 以 ELISA 測定血清中 HBsAg 的表現量.........48
結論...........................................50
未來工作.......................................52
參考文獻.......................................53
附錄...........................................63
附錄一. pEGFP-N1...............................63
附錄二. 製備Ficoll 溶液........................64
附錄三. 製備electroporation medium ............65
附錄四. 不含限制脢切位的引子(primer)...........66
附錄七. Major HBsAg gene.......................67
附錄八.插入片段與NCBI 資料庫的比對結果.........68
自述...........................................70
                                    

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