臺灣博碩士論文加值系統

猪流行性下痢病毒（porcine epidemic diarrhea virus, PEDV）於1971年在歐洲首度被發現，臺灣過往的疫情屬於傳統型PEDV零星的地區性流行，而2013年底爆發之疫情則源自新強毒變異株，感染新生仔豬出現嚴重下痢及嘔吐等臨床症狀，致死率可高達100%。檢視PEDV感染腸道細胞途徑，係與唾液酸及胺基肽酶N（aminopeptidase N, APN）可能之受體結合接觸細胞，受感染細胞逐漸且持續的壞死及脫落，並進一步造成絨毛結構萎縮，使病仔豬無法攝取所需營養、水分和礦物質而漸漸失重，最後死亡。PEDV對臺灣養豬產業造成重創，日本、韓國、中國及美國等養豬國家亦蒙受重大經濟損失，迄今仍無有效疫苗可遏止PEDV肆虐。本研究應用CRISPR/Cas9（clustered regularly interspaced short palindromic repeat/CRISPR associated protein）基因編輯技術，產製N-glycolylneuraminic acid （Neu5GC）型唾液酸合成酵素（CMP-N-glycolylneuraminic acid hydroxylase, CMAH）基因剔除（knockout, KO）豬隻，測試其後裔仔豬對PEDV之耐受性。研究團隊以CMAH 基因之CRISPR 兩個single guide RNA（sgRNA）及Cas9 mRNA顯微注射至受精卵細胞質，獲得四頭活仔L667-02、-10、-11及-12均為雙染色體CMAH基因突變，組織經檢測無Neu5GC型唾液酸存在，其子代亦同。本研究以三頭初代CMAH基因剔除母豬（L667-10,-11,-12）與其全或半同胞公豬L667-02配種，所產下F1代KO型仔豬，與同年齡WT型之新生仔豬進行接種病毒試驗；試驗一及三使用2日齡仔豬，試驗二則為3日齡仔豬，進行口腔接種1x103 TCID50/10 mL 之PEDV變異株（nv-PEDV）稀釋液，試驗期間每4 h觀察其臨床症狀及餵食嬰兒奶粉調配乳或母豬乳，同時對其活力狀態評分並記錄之；並在72 hpi（hours post inoculation）後中止試驗進行剖檢，取空腸前、中段及迴腸組織經H&E染色及免疫螢光染色分析感染病變程度。試驗結果顯示，所有仔豬皆出現下痢、嘔吐及體重減輕等典型症狀。試驗一在試驗期間餵食商業用嬰兒奶粉，6頭KO型仔豬死亡4頭（67%），WT型仔豬則6頭全數死亡（100%），兩者之存活率未有顯著差異，但腸道組織病變程度由輕到重變異極大，KO型仔豬明顯優於WT型仔豬；然試驗二使用3日齡仔豬，試驗期間同樣餵食嬰兒奶粉，則未能獲得預期與試驗一相同之結果，在存活率及腸道病變程度之比較皆無顯著差異；試驗三為釐清是否母豬乳與商業用嬰兒奶粉所含不同量之Neu5GC及Neu5AC（N-acetlylneuraminic acid）干擾試驗結果，改為餵飼試驗仔豬原對應母豬乳或脫脂乳一天，後續改餵食含5%葡萄糖乳酸林格氏液，結果顯示KO型仔豬與WT型仔豬整體存活率極高，但兩者之間並無顯著差異；經高效液相層析法分析，雖然KO型母豬乳未含Neu5GC，WT型母豬乳與商業用嬰兒奶粉，亦僅含有少量Neu5GC，且含量幾乎相同，顯示餵食不同乳源可能非干擾試驗主要因素，但試驗三餵食方法使試驗豬隻幾乎全數存活，可作為田間豬場對患病仔豬施行支持療法之範例。綜合本研究之結果，顯示對CMAH基因進行基因編輯使其未表現Neu5GC型唾液酸之豬隻無顯著改善耐受PEDV感染之特性，但有減輕嚴重性及延緩臨床症狀之現象。

Porcine epidemic diarrhea virus (PEDV) was first recognized in Europe since 1971, outbreaks of the disease also occurred in Taiwan at the end of 2013. Pigs infected by PEDV have been detected clinical sign including diarrhea and vomiting, and the morality rate of PEDV is up to 100% in neonatal piglets. The whole process of the infection of virus, first virus which bind with putative receptors such as sialic acid and aminopeptidase N(APN) to contact cells, leading infected cells gradually and continuously into necrosis, shedding and villi atrophy. Then atrophy causes the infected piglets uncapable absorbing nutrients, water and minerals, resulting in losing body weight and death. PEDV has resulted in significant economic losses in pig production in Taiwan and also in Japan, Korea, China and America, and furthermore still no effective vaccines against PEDV infection. The purpose of this study was to exam PEDV resistance of CMAH mutant (knockout, KO) piglets which generated by CRISPR/Cas9 gene editing techniques. Cytoplasm of pronuclear porcine new fertilized eggs were microinjected with two sites of sgRNA/Cas9 mRNA, and four alive piglets (L667-02,-10,-11 and -12) were born and all bi-alleles mutants. Their tissues were screened and shown without Neu5GC, and also germline transmission to their offspring. All piglets used in this study were neonatal F1 CMAH KO offspring, which delivered by the three female founders (L667-10,-11,-12) served by the male founder (L667-02). The 2-day-old or 3-day-old KO piglets were used for Exp. I and Exp. III or Exp. II, respectively; and the concurrently same age wild type (WT) piglets were used as control. Piglets were oral inoculated with 1 mL of 1x103 TCID50/mL PEDV. Then at every 4 h for three days, hand-feed milk and clean water were offered and their clinical signs were recorded for vitality analysis. After 72 h PEDV challenged, all piglets were sacrificed for necropsy, and the upper and middle of jejunum and ileum were sampled for immunofluorescence assay (IFA). Results showed that all of the piglets appeared typical clinical signs including diarrhea, vomiting and losing weight. In Exp. I, four of six KO piglets and all of six WT piglet died (67% vs. 100%), survival rate didn’t have a significant difference between two genotypes of piglets though the high variation in the extent of pathologic changes. The KO piglets apparently have a better resistance to PEDV than WT piglets. Nevertheless, in Exp. II, resulted no significant difference in clinical data, similar to Exp. I, and even in pathologic information. In Exp. III, in order to rulling out the interferece of Neu5GC/Neu5AC in sow milk and commercial baby milk powder, which used in Exp. I and II, piglets were fed dam’s milk or skim milk for one day, and then replaced to lactated Ringer’s solution supplementary with 5% glucose. Results indicated that the survival rate between both genotypes of piglets were all improved and no significant difference. Moreover, after analyzing by HPLC, commercial baby milk powder and WT dam’s milk didn’t contain a high quantity of Neu5GC, which not appeared in KO sow’s milk. The results suggest that milk isn’t the key factor in interfere the result. Furthermore, orally offer made almost the piglets survive that could be an example for supportive play chotherapy on infected piglets. This study demonstrates that CMAH gene editing, piglets without Neu5GC expression have not increased resistance to PEDV infection, but may lessen the severity of the infection and delay its occurence.

目次

中文摘要………………………………………………………………………………i
Abstract…………………………………………………………………………………iii
目次……………………………………………………………………………………v
圖次……………………………………………………………………………………vii
表次…………………………………………………………………………………viii
第一章、緒言………………………………………………………………………….1
第二章、文獻探討………………………………….………………………………....2
一、 豬流行性下痢……….….……….…….………….…….………….….…..….2
(一) 病原…………………………………………………………………......2
(二) 流行病學……………………………………………………………….…..2
(三) 感染及致病機制……………………………………………………….…..4
(四) 臨床症狀與診斷……………………………………………………….…..5
(五) 預防與治療…………………………………………………………….…..7
二、 基因編輯…………………………………………………………………...….9
(一) 鋅指核酸酶（zinc-finger nucleases, ZFNs）……………………………....…9
(二) TALENs（transcription activator-like effector nucleases）…..……………10
(三) CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence）………………………………….11
三、 基因編輯技術應用於家畜生產之成果……………………………………16
(一) 應用ZFNs產製基因編輯家畜方面………………………………………16
(二) 應用TALENs產製基因編輯家畜方面….………………………………18
(三) 應用CRISPR/Cas9產製基因編輯家畜方面…...………………………18
四、 研究目的與策略……………………………………………………………19
第三章、材料與方法……………………….……….………….…….……….……….20
一、 試驗動物…………….…….……….….…….…….….……………….…….20
(一) 實驗動物及飼養管理………………………………….……….…….…20
(二) CMAH基因剔除豬產製…………….…….……….…………………….20
(三) 配種………………………………….…………………..……………….22
(四) 分娩前準備及同期化分娩……………………….………………..……22
(五) CMAH基因型分析……………………………….………….……….23
1. DNA萃取………………………………………………………………....23
2. 溶解DNA…………………………………………………………………23
3. 聚合酶連鎖反應（Polymerase chain reaction, PCR）……………………24
4. 洋菜膠製備及電泳分析…………………………………………………24
5. 豬隻基因體DNA定序分析……………………………………………25
6. 唾液酸之高效液相層析法（high performance liquid chromatography, HPLC）分析………………………………………………………………25
（1） 試劑及標準品配置………………………………………………25
（2） 樣本處理方法與步驟……………………………………………26
二、 免疫螢光染色及評分標準….………………………………..………….26
三、 PEDV種毒製備及力價確認….……………………………..………….27
四、 仔豬PEDV攻毒試驗…………..………….…………….………….…….29
（一）試驗設計………………………………………………………………29
1. 試驗一……………………………………………………………………29
2. 試驗二……………………………………………………………………29
3. 試驗三……………………………………………………………………29
（二）場地及設施………………………………………………………………30
（三）攻毒步驟…………………………………………………………………30
（四）觀察及記錄………………………………………………………………30
（五）採樣………………………………………………………………………31
五、 統計分析…………………………………………………………………31
第四章、結果與討論……………………….…….…………….…….……….………32
一、 試驗動物………………………………………………….…….……………32
(一) CMAH 基因KO豬繁殖及基因型分析.……………………….……….32
1. 試驗一……………………………………………………………………32
2. 試驗二……………………………………………………………………32
3. 試驗三……………………………………………………………………32
二、 CMAH基因KO豬隻之組織，所含Neu5GC/Neu5AC表現分析…….….39
（一） F0之公母畜及試驗一使用仔豬組織分析……………………………39
（二） KO型、WT型母豬乳與商業用嬰兒奶粉分析………………………39
三、攻毒試驗結果…………………….………….………….………….……….43
（一）試驗一………….………….……………….………….……….……….43
（二）試驗二………….………….……………….………….……….……….49
（三）試驗三………….………….……………….………….……….……….54
第五章、總結……………………….…….………….…….…….…….……….………63
參考文獻…………………………………………………………………………….…65

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