利用噬菌體展示法製作並分析具特異性結合能力到嚴重急性呼吸道症候群冠狀病毒3a與3b蛋白的雞隻單株抗體片段

Generation and characterization of chicken antibody fragments against SARS-CoV 3a & 3b protein by phage display

鄭徹堃(Che-Kun Cheng)

指導教授 ： 呂思潔

共同指導教授 ： 楊沂淵

臺北醫學大學/醫學院/醫學科學研究所/碩士(2008年)

摘要

嚴重呼吸道症候群(Severe acute respiratory syndrome; SARS)是一個緊急的人類疾病，需要快速診斷和有效率的治療。在於不同的抗體來源，蛋黃免疫球蛋白(Immunoglobulin Y; IgY)是蛋雞(Leghorn Chicken)雞蛋中的主要抗體。IgY能夠做為研究和免疫治療之中使用的哺乳動物的抗體之外的另一種選擇。單獨表現SARS-CoV 3a蛋白便能夠促進肺細胞中的纖維蛋白表現。最近的研究也發現3a蛋白是一種新的冠狀病毒結構蛋白，可能為SARS-CoV重要的蛋白。在本實驗中，我們藉由表現2種不同的SARS-CoV的輔助蛋白(accessory protein)，分別是3a與3b蛋白，使用pET32做為vector ，以表現His tag fusion蛋白在大腸桿菌BL-21細胞中。經由Ni-NTA beads純化出帶有6個Histidine的輔助蛋白，並利用SDS-PAGE來証實，表現出來的蛋白分子量位於34.2kDa和28.1kDa之間。用這些重組的輔助蛋白來免疫萊亨蛋雞，一周免疫一次，為期一個月。並在第四次免疫後利用西方點墨法和ELISA測量抗輔助蛋白的蛋黃免疫球蛋白的抗體產生。並利用免疫螢光法證明所產生的IgY能辨認被SARS-CoV感染的細胞。進一步藉由噬菌體展示技術(phage display)的方式產生單株的蛋黃免疫球蛋白抗體，將抗體的重鏈和輕鏈DNA送入pComb3x中，並以VCSM13感染帶有質體的大腸桿菌XL-1，建立scFv的噬菌體基因庫。因此本研究希望能利用IgY作為SARS研究和檢驗治療用之抗體來源，並探討其應用於預防及治療之效果。

關鍵字

嚴重急性呼吸道症候群冠狀病毒 ； 噬菌體展示法

並列摘要

Severe acute respiratory syndrome (SARS) has been a newly emerged human disease. An effective method for rapid diagnosis or treatment of SARS should be developed. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. The expression of SARS-CoV 3a alone can up-regulate the expression of fibrinogen in lung cells. A recent study showed that 3a is a novel coronavirus structural protein suggesting that 3a may be important to SARS-CoV. In this study, truncated SARS-CoV 3a and 3b proteins were fused with histidine and expressed in the E. coli BL-21 cells. The purities of these expressed proteins were verified using SDS-PAGE. The protein molecular weights are 34.2kDa and 28.1kDa. The Leghorn chickens were immunized by intramuscular injection with purified SARS-CoV 3a or 3b fusion protein. After 4 times of challenges, high titer of anti-3a and anti-3b protein antibodies were found in the eggs of immunized chicken as demonstrated by the Western blot and Enzyme-linked immunosorbent assay. The specific binding ability of these IgY antibodies to SARS-CoV-infected cells were demonstrated by immunofluorescence assay. Furthermore, scFv (single-chain variable fragment) was expressed on the phage surface by cloning the heavy chain and light chain into pComb3X vector and then infected the XL1-Blue E.coli with VCSM13 phage. A phage displaying scFv library was also established from spleen B cells of immunized chicken. Generation of scFv antibody with specific binding ability to 3a and 3b protein and SARS-CoV infected cells is still under investigation which may be helpful in the development of therapeutic agents in the future.

並列關鍵字

SARS-CoV ； phage display

參考文獻

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