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  • 學位論文

Producing Recombinant Hemagglutinin Protein of H5N1 Avian Influenza Viruses in Chinese Hamster Overy (CHO) Cells Using Dihydrofolate Reductase and Dihydrofolate Reductase-RNA Interference

利用中國倉鼠卵巢(CHO)細胞與二氫葉酸還原酶及其核醣核酸干擾表現H5N1禽流感血球凝集素重組蛋白

指導教授 : 吳夙欽
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摘要


中文摘要 在全球各地每年都有人因感染流行性感冒而生病或是死亡,感染人類的流感病毒主要是由A型流感病毒所造成,而近年來高致病性的禽流感病毒株爆發,更可能會引起普遍性的大流行,根據世界衛生組織統計,近年來禽流感的致死率高於60%。在過去六十年之內,疫苗在對抗禽流感病毒入侵算是最有效的方法;禽流感病毒膜表面上有兩個醣蛋白,分別是Hemagglutinin(HA)與Neuraminidase(NA),HA蛋白是禽流感病毒與人體細胞結合的主要接收器,並且也是最主要能夠產生強力的免疫反應的蛋白,在本篇實驗中,我在中國倉鼠卵巢細胞中表現序列最佳化的禽流感H5HA蛋白與GCN4結合,幫助重組的蛋白分泌出細胞外。 中國倉鼠卵巢細胞和二氫葉酸還原酶基因增幅系統已經被廣泛應用在生技製藥產業,用於生產穩定的中國倉鼠卵巢細胞株,用以製造重組的藥用蛋白。本篇研究使用了在基因增幅選擇法中加入以dhfr為標的之RNA干擾載體sd2與驅動dhfr的弱啟動子pHSV-TK去改善並選擇具有HA蛋白高度生產力的中國倉鼠卵巢細胞株;首先,由弱啟動子pHSV-TK驅動所能轉錄出的RNA量較少,並且dhfr的RNA干擾性載體sd2可使dhfr mRNA被切開,因此dhfr的細胞複製數被迫需增加,進而去提高在dhfr基因表現缺失的中國倉鼠卵巢細胞中,在dhfr下游的重組流感HA蛋白表現量提升,並希望以生產出大量醣化後的流感HA蛋白當作次單位疫苗來針對近年來流行的禽流感做有效的防範。結果發現以RNA干擾性載體sd2與不論是強啟動子或弱啟動子的dhfr基因載體一起使用是比只有單一使用弱啟動子的dhfr基因載體來的好。

並列摘要


Abstract Influenza infection cause human morbidity and death worldwide in every year. Infectous human influenza virus is major formed by influenza A virus. High pathogenic avian influenza viruses perhaps cause outbreak of pandemic, recently. According to World Health Organization estimates, the mortality of H5N1 in humans is greater than 60%. Over the past 60 years, vaccination has been the most effective method to protect the population against avian influenza virus infection. Hemagglutinin (HA), a glycoprotein on the surface of influenza virus, is responsible for major receptor binding to human cells, and mainly as a target for vaccine development. Chinese Hamster Ovary cells (CHO) and dihydrofolate reductase (dhfr)/methotrexate (MTX) gene amplification system are commonly used in biopharmaceutical industry to generate stable expressing recombinant proteins CHO cell clones. In this study, I expressed the optimizing H5HA protein coding sequence conjugated with GCN4 in CHO cells to produce secreted recombinant HA proteins. During gene amplification, we used dhfr-targeting RNA silencing vector (sd2) and Herpes simplex virus thymidine kinase weak promoter (pHSV-TK-driven dhfr) to weaken DHFR expression for improvement of high producer CHO cell clones selection. The high producer cell clones with sd2 and pHSV-TK-driven dhfr would be selected and could be used for mass producing recombinant HA glycoprotein subunit vaccine. These studies suggest that the incorporation of psd2 silencing vector with either strong or weak promoter vectors were better than the use of weak promoter vector only.

參考文獻


Assaraf, Y.G., Schimke, R.T. (1987). Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry. Proc Natl Acad Sci U S A Oct;84(20):7154-8.
Backliwal, G., Hildinger, M., Chenuet, S., Wulhfard, S., De Jesus, M., Wurm, F. M. (2008). Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36(15):e96.
Bebbington, C.R., Renner, G., Thomson, S., King, D., Abrams, D., Yarranton, G.T. (1992). High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (N Y) Feb;10(2):169-75.
Bell, A.C., Felsenfeld, G. (1999). Stopped at the border: boundaries and insulators. Curr Opin Genet Dev. Apr;9(2):191-8.
Benton, T., Chen, T., McEntee, M., Fox, B., King, D., Crombie, R., Thomas, T.C., Bebbington, C. (2002). The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein. Cytotechnology Jan;38(1-3):43-6.

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