利用胡蘿蔔表達禽流感病毒血球凝集素基因之研究

禽流感 (avian influenza) 是由屬於正黏液病毒科 (Orthomyxoviridae) 之禽流感病毒所引起。目前禽流感疫苗多為以雞胚胎進行病毒增殖之死毒疫苗，製作費時。植物疫苗 (plant-based vaccine) 具有容易大量生產、成本低等優點，其中又以葉綠體基因轉殖植物具有高度表達外源基因、無基因靜默等諸多優點，可作為生產醫藥蛋白之良好途徑。為了開發較低成本又可大量生產之禽流感疫苗生產平台，先前已將可誘發宿主免疫反應之禽流感病毒血球凝集素 (hemagglutinin, HA) 基因，轉殖至胡蘿蔔葉綠體基因組中表達，經抗生素篩選後，以綠光照射擬轉殖系癒傷組織、根部及葉片，可觀察到報導基因DsRed之表現。經PCR分析HA擬轉殖株，可見合成ha基因之目標條帶；以西方轉漬法 (Western blot) 分析，可在65 kDa處有代表目標蛋白之條帶，表示HA蛋白可於胡蘿蔔葉綠體中表達。為了使HA轉殖之胡蘿蔔葉綠體均質化，將轉殖株葉片逆分化為癒傷組織後再生數循環，過程中持續於含有spectinomycin之培養基中進行篩選，以獲得均質之轉殖系。另將作為免疫增強佐劑之大腸桿菌忌熱毒素 (heat-labile enterotoxin B subunit, LTB) 基因，及LTB基因連結HA基因之兩種載體，以農桿菌轉殖法及基因槍轉殖法分別轉殖至胡蘿蔔核基因組及葉綠體基因組中。再生之核基因轉殖LTB-HA及LTB癒傷組織經由GUS活性組織化學染色分析，均有GUS活性。葉綠體轉殖 LTB-HA之再生癒傷組織、根部及葉片於藍光照射下可見GFP之螢光訊號；以專一引子進行PCR分析，可合成ha基因之目標條帶。為了大量生產HA蛋白，以LTB-HA轉殖之癒傷組織進行懸浮培養。培養基中添加proline之懸浮細胞生長速度及增重倍率較添加glutamine 及控制組高，可收穫較多之HA蛋白。

關鍵字

禽流感；植物疫苗；葉綠體基因轉殖；胡蘿蔔

並列摘要

Avian influenza is caused by avian influenza virus which belongs to Orthomyxoviridae. The current virus-inactivated vaccines for avian influenza are produced in chicken embryos and time-consuming. High expression level of foreign protein and no gene silencing effect make chloroplast transformation plants as an ideal system to produce plant-based vaccines, which has low production cost and is easy to scale up. To develop an effective production system for avian influenza oral vaccine, ha gene has been transformed into carrot chloroplast genome by biolistic method previously. Under green light, the red fluorescence produced by reporter gene DsRed was observed in callus, roots and leaves of putative transgenic carrot lines. HA transplastomic lines were confirmed by PCR analysis, and protein expression was investigated by Western blot analysis showing a target band of 65 kDa. In order to obtain homoplastomic plants, leaf of HA transplastomic carrot was conducted to dedifferentiate into callus for several cycles. On the other hand, adjuvant protein E. coli heat-labile enterotoxin B subunit (LTB) gene, and LTB linked to ha gene were transformed into carrot nucleus and chloroplast genome by Agrobacterium-mediated and biolistic method, respectively. Both nucleus-transformed LTB and LTB-HA calli showed positive results in GUS staining. The green fluorescence produced by reporter gene GFP was observed in LTB-HA chloroplast transformed callus, leaves and roots. After transgene integration confirmed by PCR analysis, LTB-HA transplastomic calli were used as the material to establish a suspension culture system for large scale production of plant-derived HA protein. Growth rate and proliferation fold of suspension cells reached the highest in medium containing prolineresulting in higher protein yield of HA protein.