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研究生:謝佩珍
研究生(外文):Pei-Chen Hsieh
論文名稱:應用酵母菌Pichiapastoris表現系統生產豬瘟病毒醣白E2及其抗原性之分析
論文名稱(外文):Production of the classical swine fever virus glycoprotein E2 by Pichia pastoris expression system
指導教授:黃千衿
指導教授(外文):Chienjin Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:85
中文關鍵詞:豬瘟病毒醣蛋白酵母菌真核表現系統
外文關鍵詞:Classical swine fever virusGlycoproteinYeast eukaryotic expression system
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豬瘟是一種豬的重要傳染病,其病原為豬瘟病毒(classical swine fever virus, CSFV)。豬瘟病毒外層封套上具有3種醣蛋白,分別是Erns、E1及E2,而其中的Erns和E2已被證實可誘發宿主產生中和抗體。本實驗之目的即嘗試利用酵母菌Pichia真核表現系統來表現E2醣蛋白,以提供為發展豬瘟次單位疫苗的材料。首先利用聚合酵素鏈鎖反應(polymerase chain reaction, PCR)及TA cloning方法進行豬瘟病毒E2醣蛋白基因的選殖,經DNA定序確定正確序列後再將E2基因片段選殖於具有α-factor分泌序列的酵母菌Pichia表現載體pGAPZαC,而構築出重組質體pE2 6-2G用於轉形。經轉形作用(transformation)後成功篩選出具抗Zeocin且含有E2基因之重組P. pastoris酵母菌株。萃取重組菌株之染色體DNA並以E2基因之特異性引子進行PCR檢測,結果顯示其染色體上均含有E2基因。以抗豬瘟病毒醣蛋白E2之特異性單源抗體進行西方轉漬分析(Western blotting)之結果顯示抗體可特異性辨認酵母菌所表現之E2蛋白﹔進一步以表現之E2蛋白免疫小白鼠後,所收集之免疫血清在間接免疫螢光分析(IFA)試驗中,亦可特異性辨認豬瘟病毒所感染之細胞,顯示此表現之E2蛋白具有正確之抗原性。
Classical swine fever (CSF) is an important infectious disease of swine, its causative agent is Classical swine fever virus (CSFV). The virion is surrounded by an envelope containing three viral glycoproteins, Erns (E0) , E1 as well as E2 both Erns and E2 have been shown to be capable of inducing neutralizing antibodies in host. The purpose of this study is to produce the CSFV glycoprotein E2 amount in the yeast Pichia pastoris eukaryotic expression system and furtherly to developing an efficient CSFV subunit vaccine. Glycoprotein E2 gene was cloned by polymerase chain reaction (PCR) cloning strateg and then subcloned into the P. pastoris expression vector pGAPZαC to generate the recombinant plasmid pE2 6-2G. After transformation of P. pastoris, the recombinant strains resisting to high concentration of the antibiotic Zeocin were selected. The chromosome DNA of each transformant was extracted and further identified to contain the CSFV E2 gene by PCR analysis. The expressed E2 proteins in culture supernatant and cellular lysates of recombinant P. pastoris were analyzed by SDS-PAGE after 96h incubation, and the major 55 kDa secretary protein was specifically recognized by the monoclonal antibody against the E2 protein. Furthermore, immunostaining of the CSFV-infected cells using mouse serum immuned with the expressed E2 protein in indirect fluorescent antibody (IFA) assay revealed specifically cytoplasmic staining.
中文摘要 ------------------------------------------------- I
英文摘要 ------------------------------------------------- II
目錄 ----------------------------------------------------- III
表次 ----------------------------------------------------- IV
圖次 ----------------------------------------------------- V
第一章 緒言 --------------------------------------------1
第二章 文獻探討 ----------------------------------------3
第一節 酵母菌真核表現系統 ------------------------------3
第二節 嗜甲醇酵母菌Pichia pastoris ---------------------4
第三節 Pichia pastoris之蛋白質後修飾作用 ---------------10
第四節 豬瘟與豬瘟病毒 ----------------------------------12
第五節 豬瘟之研究現況 ----------------------------------19
第三章 材料與方法 --------------------------------------21
第一節 表現載體之構築 ----------------------------------21
第二節 酵母菌之轉形 ------------------------------------23
第三節 轉形酵母菌株之確認 ------------------------------24
第四節 重組酵母菌之表現 --------------------------------26
第五節 重組酵母菌表現產物之分析 ------------------------27
第四章 結果 --------------------------------------------29
第五章 討論 --------------------------------------------44
參考文獻 -------------------------------------------------47
附錄 -----------------------------------------------------61
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