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研究生:張熙展
研究生(外文):Shi-Jan Chang
論文名稱:豬瘟病毒之E2重組次單位抗原之劑量決定試驗及其單株抗體之備製
論文名稱(外文):Dose determination of recombinant subunit CSFV E2 antigen and preparation of monoclonal antibody to E2
指導教授:簡茂盛簡茂盛引用關係
指導教授(外文):Maw-Sheng Chien
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:86
中文關鍵詞:豬瘟病毒E2醣蛋白單株抗體
外文關鍵詞:classical swine fever virusglycoprotein E2monoclonal antibodies
相關次數:
  • 被引用被引用:2
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豬瘟為一高接觸性與高致死性之法定傳染病,常造成養豬產業重大的經濟損失。而豬瘟病毒之E2醣蛋白為誘導宿主產生中和抗體之主要抗原,本研究則嘗試以桿狀病毒所表現之豬瘟病毒E2次單位蛋白,應用於豬隻進行不同抗原劑量之免疫效力試驗。實驗首先分別以10、20、40、60、80和100 μg等不同劑量之E2重組次單位抗原(rE2)免疫豬隻,顯示所有免疫組豬隻其中和抗體力價均有揚升之情形,且於免疫10 μg之rE2組即可誘發豬隻產生具保護力價之免疫反應。另外,免疫高劑量抗原組(100 μg rE2)於注射部位並無產生任何不良反應且豬隻在臨床上亦無任何病徵,顯示此疫苗對豬隻具高度安全性。經選擇低劑量組(10 μg和20 μg rE2)之免疫組豬隻以豬瘟病毒進行攻毒試驗,並以其臨床症狀、體溫、增重率、白血球總數、血清學、RT-PCR、肉眼病變和組織病理學等項目進行評估與分析,其結果顯示免疫20 μg E2重組疫苗組於攻毒後均存活,且無呈現明顯豬瘟之臨床症狀。進一步於分子檢測上,經20 μg rE2免疫組豬隻經攻毒後再以RT-PCR偵測各臟器之病毒核酸之分佈情形,結果顯示不論於血液、淋巴器官和腦部等樣本均無發現病毒核酸之存在。上述結果顯示豬隻經免疫20 μg E2重組抗原後的確可誘發宿主產生足夠的中和抗體來抵抗豬瘟病毒之感染。
另一方面,本試驗亦藉由融合瘤細胞技術以嘗試製備辨識豬瘟病毒之單株抗體,經利用純化之重組E2蛋白多次免疫BALB/c小鼠後,再分離其脾臟細胞並與NS-1骨髓瘤細胞進行融合,再應用酵素結合免疫吸附法和間接螢光抗體染色法篩選陽性細胞,目前有10株融合細胞株呈現陽性反應。
Classical swine fever (CSF) is a highly contagious and fatal viral disease of pigs that causes considerable economic loss in swine industry. Glycoprotein E2 is the most immunogenic determinant of the CSFV that induces neutralizing antibody against CSF infection in swine. The aim of this study was to determine the appropriate dose of baculoviral expressed recombinant CSFV E2 (rE2) that can elicit protective immunity against CSFV. The various concentration of rE2 antigen as 10、20、40、60、80 and 100 μg was applied for immunization in pigs respectively and all vaccined animal were able to elicit good protective immunity. In addition, no adverse side effect was noticed after immunization of high dose of CSFV rE2 protein (> 80 μg rE2), indicating non-toxicity and safety of rE2 in pigs. The result indicated that pigs vaccinated with 10 μg or above of rE2 can induce effective antibody titer against CSFV. Furthermore, lower vaccination trials (10 μg and 20 μg of rE2 immunized group) were challenged with virulent CSFV in order to evaluate the vaccine efficacy according to clinical score, body temperature, weight increasing rate, white blood cell count, serological analysis, RT-PCR, gross lesion and histopathological examination. These data showed that all pigs vaccinated with 20 μg of rE2 survived with no typical clinical signs after CSFV challenge. Moreover, no viral RNA can be detected in 20 μg of rE2 immunization group in blood, lymphatic organs and brain by RT-PCR. The results indicated that pigs vaccinated with 20 μg of rE2 were sufficient to elicit good protective immunity against CSFV. In addition, we also tried to prepare monoclonal antibodies against CSFV by hybridoma technique. BALB/c mice were immunized with purified rE2 proteins and then the splenocytes were isolated and fused with NS-1 myeloma cells. Ten clones of hybridoma cell line were obtained at this moment after screening using ELISA and IFA methods.
目錄
頁次

中文摘要--------------------------------------------------------------------------------- I
英文摘要--------------------------------------------------------------------------------- II
目錄--------------------------------------------------------------------------------------- III
表次--------------------------------------------------------------------------------------- VI
圖次--------------------------------------------------------------------------------------- VII
第一章 前言--------------------------------------------------------------------------- 1
第二章 文獻回顧--------------------------------------------------------------------- 3
第一節 豬瘟之歷史背景------------------------------------------------------ 3
第二節 流行病學--------------------------------------------------------------- 4
第三節 臨床症狀--------------------------------------------------------------- 5
第四節 肉眼變化--------------------------------------------------------------- 5
第五節 組織病理學變化------------------------------------------------------ 6
第六節 致病機轉--------------------------------------------------------------- 7
第七節 豬瘟病毒之理化特性和結構形態--------------------------------- 9
第八節 豬瘟病毒基因型之研究--------------------------------------------- 10
第九節 豬瘟病毒蛋白之研究------------------------------------------------ 12
第十節 抗體之生產------------------------------------------------------------ 16
第十一節 培養液篩選原理----------------------------------------------------- 17
第十二節 豬瘟之研究現況---------------------------------------------------- 19
第三章 材料與方法------------------------------------------------------------------ 20
第一節 抗原之備製-------------------------------------------------------------- 20
一、 E2重組蛋白之大量生產------------------------------------------ 20
二、 E2重組蛋白濃度測定--------------------------------------------- 21
第二節 小鼠免疫試驗----------------------------------------------------------- 22
一、 免疫-------------------------------------------------------------------- 22
二、 血清抗體力價分析-------------------------------------------------- 22
第三節 豬隻免疫試驗----------------------------------------------------------- 23
一、 免疫用之抗原製作-------------------------------------------------- 23
二、 豬隻來源及組別----------------------------------------------------- 23
三、 血清抗體力價分析-------------------------------------------------- 24
第四節 豬隻攻毒試驗----------------------------------------------------------- 24
一、 攻毒流程-------------------------------------------------------------- 24
二、 攻毒後檢查項目----------------------------------------------------- 25
三、 血清抗體力價分析---------------------------------------------------25
四、 逆轉錄鏈聚合反應-------------------------------------------------- 25
第五節 抗原之純化-------------------------------------------------------------- 28
一、 備製結合抗體之Sepharose---------------------------------------- 28
二、 E2重組蛋白之純化------------------------------------------------- 28
三、 E2重組蛋白之定量及保存---------------------------------------- 29
第六節 單株抗體之備製-------------------------------------------------------- 29
一、 免疫------------------------------------------------------------------- 29
二、 骨髓瘤細胞之培養------------------------------------------------- 30
三、 細胞融合------------------------------------------------------------- 30
四、 陽性細胞株之篩選------------------------------------------------- 31
五、 融合細胞之單株化------------------------------------------------- 33
六、 單株抗體之生產---------------------------------------------------- 34


第四章 結果--------------------------------------------------------------------------- 35
第一節 E2重組蛋白表現及抗原性分析------------------------------------- 35
一、 E2重組蛋白表現之情形------------------------------------------ 35
二、 E2重組蛋白抗原性分析------------------------------------------ 35
第二節 E2重組蛋白免疫試驗------------------------------------------------- 35
一、 小鼠免疫試驗-------------------------------------------------------- 35
二、 豬隻免疫試驗-------------------------------------------------------- 36
第三節 攻毒試驗----------------------------------------------------------------- 36
一、 攻毒試驗後其實驗豬之臨床表徵-------------------------------- 36
二、 血清學分析----------------------------------------------------------- 38
三、 肉眼病理學檢查----------------------------------------------------- 38
四、 組織病理學檢查----------------------------------------------------- 38
五、 逆轉錄鏈聚合反應-------------------------------------------------- 39
第四節 單株抗體之備製-------------------------------------------------------- 40
一、 抗原之純化----------------------------------------------------------- 40
二、 免疫後之血清測定-------------------------------------------------- 40
三、 細胞融合之結果----------------------------------------------------- 40
四、 陽性細胞株之篩選-------------------------------------------------- 41
五、 極限稀釋-------------------------------------------------------------- 41
第五章 討論--------------------------------------------------------------------------- 42
第六章 文獻參考--------------------------------------------------------------------- 73
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