利用新穎桿狀病毒多效表現載體發展流感疫苗之研究

Development of flu vaccine by a novel polycistronic baculovirus expression vector

羅雅雯(Ya-Wen Lo)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2010年)

https://doi.org/10.6840/cycu201000512

摘要

屬於正黏膜病毒科 (Orthomyxoviridae) 的流感病毒一直是公共衛生的重要課題，例如1918年的H1N1西班牙流感病毒與2009年H1N1新流感。其中，最受矚目的莫過於高病原性的H5N1禽流感病毒 (Hight pathogenic avain influenza virus, HPAI)。由於H5N1禽流感病毒會經由鳥類的遷徙而造成大流行，受感染的雞隻死亡率近乎100%。不但造成家禽業巨大的損失，亦有引發高致死率人類流感的風險，因此禽流感疫苗的研發是一極受重視的研究領域。先前已雞隻喉頭氣管炎病毒 (infectious laryngotracheitis, ILTV) 所進行的DNA疫苗研究顯示，同時接種帶有HA (Hemaglutinin) 及 NA(Neuraminidase) 抗原之雞隻其保護效果較單只有單獨接種HA或NA抗原疫苗來的好，因此本論文即構築同時帶有紅血球凝集素HA及神經胺酸酶NA多效桿狀病毒載體vAc-NA-EGFP-Rhir-HA並分析於Sf21細胞及擬尺蠖蟲體之HA及NA抗原的表現量，以評估發展禽流感疫苗的可行性，利用西方墨點法、NA酵素活性測定及免疫螢光染色法證實vAc-NA-EGFP-Rhir-HA此多效載體可成功於Sf21細胞及擬尺蠖共表現HA及NA蛋白，期望此利用新穎的桿狀病毒多效表現載體的禽流感次單位疫苗未來在動物試驗中，HA及NA蛋白可一起誘導免疫反應達到較佳的保護之效果。

關鍵字

禽流感 ； 桿狀病毒

並列摘要

Since 2003, outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in human and poultry, that is Orthomyxoviridae. H5N1virus lead to generalized infections in chickens with mortality rates close to 100%. At present, there are over hundreds of millions of birds and several hundred people died and cause significantly economic losses in worldwide. Avian influenza virus have two glycoproteins on viral surface, hemagglutinin (HA) and neuraminidase (NA). HA is the major immunogen on the envelope of avian influenza virus, and it is the main target antigen of the humoral immune response in host animals. NA facilitates viral release from cells by removing sialic acid from sialyloligosaccharides on surface of cells and virals. In response to the pandemic disease by H5N1 influenza virus, the flu vaccine development is imperative. Commercial vaccines come from ckicken embryos, which is time-consuming and the cost is too high for poultry. In this study, a novel tricistronic baculovirus expression vector was generated and was used to express NA, HA gene and a convenient traceable fluorescent reporter gene, EGFP. The tricistronic transcript containing ORFs of NA, HA and EGFP is controlled by the polyhedron promoter. To express the three genes simultaneously, NA was translated through a cap-dependent mechanism, while the EGFP and HA were translated by the IRESs derived from Perina nuda virus (PnV) and the 5' UTR of Rhopalosiphum padi virus (RhPV), respectively. We conducted the following experiment to proof the correctly expression of the tricistronic baculovirus vector in Sf21 cells and T. ni larvae. First, the recombinant baculovirus, vAc-NA-EGFP-Rhir-HA, can be identified by the green fluorescence under fluorescence microscope. Second, the vAc-NA-EGFP-Rhir-HA infected Sf21 cells and T. ni larvae revealed the neuraminidase enzyme activity. Finally, Western blot and with anti-HA monoclonal antibody indicate that the third ORF, the HA was expressed. And immune flouracence assay confirned NA and HA expressed in Sf21 cells. Thus, the tricistronic recombinant baculovirus, vAc-NA-EGFP-Rhir-HA, can expressed the NA and HA proteins simultaneously in Sf21 insect cells. We hypothesize that the HA and NA combined vaccine formula produced by this vAc-NA-EGFP-Rhir-HA tricistronic vector can be used as an effective for poultry, the HA will induce the antibodies that against the flu virus entry route and the NA will induce the antibodies that prevent the flu viruses releasing.

並列關鍵字

NA ； HA ； baculovirus

參考文獻

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