桿狀病毒生產基因工程蛋白及其功能研究

Production and functional analysis of engineered proteins using baculovirus expression system

林怡婷(Lin Yi-ting)

指導教授 ： 趙裕展

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2005年)

摘要

利用桿狀病毒生產系統來生產基因工程蛋白，可以生產出大量欲研究未知功能蛋白，來瞭解其生物功能和活性。在本篇論文中使用兩種桿狀病毒生產系統，即AcMNPV和BmNPV，來生產豬的乳鐵蛋白、大腸桿菌的植酸酵素及蜂王漿中的一種小蛋白，apisimin。使用BmNPV生產系統的目的是為了日後可以在家蠶中大量生產並應用於豬的飼料添加物中來增強豬隻的抵抗力和減少排泄物的污染。在本篇論文中，以成功的在Ac和Bm系統生產出豬的乳鐵蛋白、大腸桿菌的植酸酵素和apisimin，並純化出apisimin來做相關實驗以瞭解生物活性。以桿狀病毒生產系統所生產出的植酸酵素，其酵素活性比用植物生產的要來的高; 在apisimin的生物活性試驗上，在免疫方面，對細胞沒有毒性，由ELISA的結果中發現對於TNF-?、IL1-?和IL-6的量有隨著濃度增加而增加，可以活化免疫反應; 在急性發炎反應下，經由LPS刺激後TNF-a隨著濃度增加而下降，但是在IL1-?和IL-6表現上，卻有隨著濃度增加而先升高再下降，由此判斷apisimin可能具有雙向調節的功能。在抗菌方面，在測試的11種菌株中並沒有發現有抑制細菌的功能，但是從清除自由基的能力來看，apisimin有清除自由基的能力，且有隨著濃度增加而上昇。由以上可以得知利用桿狀病毒生產外源蛋白，所生產出的蛋白是具有生物活性的，對於研究未知功能的蛋白是一個很好的工具。

關鍵字

桿狀病毒 ； 豬乳鐵蛋白 ； 植酸酵素 ； 蜂王漿

並列摘要

Baculovirus expression system is a conventional tool for production of large amounts of engineered proteins, which may be subsequently purified for further studies in their biological and enzymatic functions. In this study, two baculovirus expression systems, AcMNPV and BmNPV, were used to produce 3 recombinant proteins: porcine lactoferrin, phytase of E. coli, and apisimin, which is a small component of royal jelly. The use of BmNPV expression system is for subsequent massive production of recombinant protein in silkworm, which, after purification, will be added in feeding to increase porcine immunity and alleviate pollutions caused by porcine feces. We’ve successfully produce recombinant porcine lactoferin, phytase of E. coli, and apisimin of royal jelly by AcMNPV and BmNPV expression systems. Recombinant apisimin protein was further purified for studies for its biological and enzymatic activities. Purified phytase protein produced by baculovirus expression system exhibited higher enzymatic activity compared to phytase produced by plant. Purified apisimin protein produced by baculovirus expression system did not cause cell toxicity in immunological experiment. From ELISA experiment, productions of TNF-?, IL-1-? and IL-6 increased proportionally with the concentration of apisimin, indicating activation of immune responses by apisimin. Under inflammatory condition, e.g., stimulation by LPS, production of TNF-? decreased inversely with the concentration of apisimin. On the contrary, productions of IL-1-? and IL-6 show initial increase, followed by a steady decline as the concentration of apisimin increased. Therefore, it’s proposed that apisimin could regulate immune response bi-directionally. In test for antibiotic function, apisimin did not suppress bacterial growth in 11 bacteria strains tested. However, it exhibited ability in cleaning up free radicals, and this function was concentration-dependent. In conclusion, purified recombinant proteins produced by baculovirus expression system were shown to be biologically functional. Therefore, baculovirus expression system is a useful tool in studying functions of proteins whose functions are still unknown.

並列關鍵字

baculovirus ； porcine lactoferrin ； phytase ； apisimin

參考文獻

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