豬丹毒桿菌莢膜抗原性基因分子選殖表現與單株抗體之製備

Cloning and Expression of the Capsule Antigenic Gene from Erysipelothrix rhusiopathiae and Preparation of the Monoclonal Antibody

鄭貴元(Kue-Wan Zen)

指導教授 ： 廖明輝

國立屏東科技大學/農學院/生物科技研究所/碩士(2008年)

https://doi.org/10.6346/NPUST.2008.00063

摘要

豬丹毒桿菌 ( Erysipelothrix rhusiopathiae ) 是一種革蘭氏陽性菌，會造成動物的丹毒症與人的類丹毒。在豬隻方面，本菌會引起急性敗血症或關節炎與心內膜炎等慢性疾病。豬丹毒桿菌的莢膜對於疾病的致病性扮演著重要角色，是導致本菌發病的因子。本實驗之目的在於利用豬丹毒桿菌莢膜多胜肽抗原性區域表現出的蛋白質。首先以蛋白質分析軟體分析及預測可能具抗原性並且為高度保留區之氨基酸區段，再設計引子以聚合酶鏈鎖反應 ( polymearase chain reaction, PCR ) 增幅，利用pET-32a表現載體進行選殖，將構築好之載體轉形至勝任細胞BL21 ( DE3 ) pLysS中，以IPTG誘導蛋白質表現。接著利用金屬螯合層析法 ( metal chelate affinity chromatography ) 將表現之蛋白質進行純化，然後分別免疫BALB/c鼷鼠四次，以生產多株抗體。再將多株抗體利用西方轉漬法 ( Western blotting )分析其是否為豬丹毒桿菌莢膜多胜肽蛋白，之後開發生產單株抗體，以供未來檢測與診斷。

關鍵字

豬丹毒桿菌 ； 莢膜 ； 抗原性 ； 單株抗體

並列摘要

Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animal and erysipeloid in humans. Erysipelas in swine can occur as an acute septicemia or chronic disease,which is characterized by arthritis and endocarditis. The capsule of Erysipelothrix rhusiopathiae may be important in the pathogenicity of the disease. The objective of this study is to identify the antigenic regions of polypeptide of capsule for development of monoclonal antibody. The amino acid sequences with high antigenicity were predicated and amplified by PCR. The correct amplicons were cloned into pET-32a vectors, transformed into competent cell [ BL21 ( DE3 ) pLysS ], and expressed after induction with IPTG. BALB/c mouse were immunized with the expressed proteins purified with HiTrap affinity columns, sera from immunized mice were used to detect the proteins in Western blots. Then, the monoclonal antibody will be development and production.

並列關鍵字

Erysipelothrix rhusiopathiae ； capsule ； antigenicity ； monoclonal antibody

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