利用雙效BacMam載體於哺乳細胞內生產禽流感H5N1血球凝集蛋白與神經胺酸

H5N1是一種具高病原性的A 型流感病毒，其主要是感染鳥類，但也有人類受到感染而死亡的例子，最初在亞洲爆發流行後來擴大至其他大陸國家。為了防止人類的H5N1流感造成全國性的傳染，發展流感疫苗是當今迫切需要的工作。考量到發展H5N1疫苗之必要性，本篇論文利用多效表現載體 (BacMam vector) 在哺乳動物細胞中同時表現紅血球凝集素蛋白 (HA)及神經胺酸苷酶 (NA)做為疫苗製劑。我們利用巨細胞病毒啟動子 (CMV promoter)表現HA蛋白及一段結合腸病毒71型5’端未轉譯區域內之644核甘酸序列 (1-645 bp) 及蚜蟲病毒 (Rhopalosiphum padi virus, RhPV) 5’端未轉譯區域 (5’ UTR)內之110核甘酸序列(309-418 bp)所組成之新穎內核醣體轉譯子 (Internal ribosome entry site, IRES)表現NA蛋白，以及利用RhPV IRES全長序列表現綠螢光蛋白 (enhance green fluorescent protein, GFP)，可用來觀測病毒感染情況 (Infection)及轉導 (Transduction)效率。此三效病毒表現載體命名為vAc-CMV-HA-EV71RP110-NA-Rhir-EGFP。本研究使用骨肉瘤細胞 (U2OS)做為病毒轉導的宿主，期待可成功生產出HA, NA以及GFP三種蛋白。因此此三效載體可提供未來發展DNA疫苗以減緩流感病毒H5N1傳染可能性。

關鍵字

BacMam ；禽流感H5N1 ；神經；胺酸

並列摘要

Outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species began in Asia and have spread to other continents. So, an urgent need to develop vaccines would protect the human population in the event of a pandemic. Considering the necessity of vaccine development against H5N1, this study researches on H5N1 influenza hemagglutinin (HA) proteins – infection factors and neuraminidase (NA) proteins - spread factors. They are simultaneously expressed in mammalian cells by the BacMam vector. It utilizes that with Cytomegalovirus (CMV) gene promoter, EV71RP110 IRES (the 5' untranslated region (5’UTR; 644 nucleotides) of Enterovirus 71 virus and RhPV IRES (the 5’UTR (110 nucleotides) of Rhopalosiphum padi virus) and RhPV (Rhopalosiphum padi virus) IRES, a tri-cistronic baculovirus expression vector (vAc-CMV-HA-EV71RP110-NA-Rhir-EGFP) includes HA, NA and Green fluorescent protein (EGFP) which can monitor the transfected or transduced mammalian cells, that should be highly expressed in mammalian cells. Mammalian cells, U2OS, would be used to express HA, NA and EGFP proteins by transfection and transduction. Therefore, the tri-cistronic plasmid could induce immune responses and effectively support to influenza DNA vaccine development as a public health mediation to reduce a pandemic of H5N1 influenza.

並列關鍵字

hemagglutinin ； neuraminidase ； BacMam ； Influenza virus H5N1

參考文獻

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利用雙效BacMam載體於哺乳細胞內生產禽流感H5N1血球凝集蛋白與神經胺酸

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