應用轉殖胡蘿蔔表達禽流感病毒血球凝集素基因之研究

Abstract

家禽流行性感冒 (Avian influenza) 簡稱禽流感，是一種發生於禽鳥間的流行性感冒，家禽中尤以雞、鴨最易受到感染，嚴重時常導致死亡，病毒傳染速度快，常造成農業上的重大損失。疫苗的研發為預防禽流感的方法之一，利用基因轉殖植物生產疫苗，不但可作為生物反應器進行大量生產而降低疫苗成本，亦可免去人類病原汙染的疑慮。 本研究先前已利用農桿菌媒介法轉殖編碼為醣蛋白之禽流感病毒表面抗原決定位 ha 基因至胡蘿蔔中，經聚合酶連鎖反應 (polymerase chain reaction) 和南方氏雜交分析可偵測到 ha 基因片段，利用酵素連結免疫吸附分析 (enzyme-linked immunosorbent assay) 可偵測到 65 kDa 之 HA 外源蛋白表達。為了提高 HA 外源蛋白表達量，進一步利用葉綠體轉殖法 (chloroplast transformation) 進行轉殖，期望提高胡蘿蔔轉殖株中 HA 外源蛋白表達量。 本研究以胡蘿蔔葉綠體中 rpoA 與 petD 基因作為側翼序列，將禽流感病毒表面抗原決定位 ha 基因構築於側翼序列之間，以基因槍法 (particle bombardment) 將構築質體轉殖至胡蘿蔔葉綠體中，以抗生素 spectinomycin 進行篩選，將具抗性之轉殖細胞進一步分析，經聚合酶連鎖反應可分析到 ha 基因片段，為確認目標基因確實轉殖至胡蘿蔔葉綠體中，利用側翼序列外之引子進行分析，亦可得到目標片段，顯示目標基因已轉殖至胡蘿蔔葉綠體中。萃取 RNA 進行反轉錄聚合酶連鎖反應 (reverse transcription polymerase chain reaction, RT-PCR) 可偵測到 ha 基因表現；進一步萃取轉殖株葉片之蛋白質，利用 HA 單株抗體進行免疫轉漬分析 (immunoblot analysis)，結果顯示轉殖株中皆含有 65 kDa 之 HA 蛋白，顯示外源基因可順利表達蛋白。

Avian influenza is an influenza in poultry flocks. Chickens and ducks are susceptible to infect (avian influenza virus, AIV) and often leading to death. The transmitted speed of the virus is very fast. AIV causes a great damages of the agriculture. Vaccine development is a strategy to prevent AIV. Transgenic plants have been used as a large-scale and low-cost production system of safe and pharmaceutical proteins. Previously ha gene has been transfered into carrots by Agrobacterium tumefaciens-mediated transformation method. The ha gene encoding the AIV surface glycoprotein (hemagglutinin, HA), a major antigen of AIV agent. The foreign ha gene was detected by polymerase chain reaction and Southern blot analysis. Enzyme-linked immunoassays revealed that HA protein expression in transgenic carrot. In order to enhance the foreign protein expression, the ha gene was further transfered into carrot chloroplasts. The rpoA and petD genes derived from carrot chloroplast genome were used as flanking sequence for plasmid construction. The ha gene encoding the AIV surface glycoprotein was constructed between the flanking sequences and transfered into chloroplasts of carrot by particle bombardment. After antibiotic selection transgenic carrot calli were regenerated into plantlets. DNA from putative transgenic plants was extracted and the integration of ha gene in carrot chloroplast genome was confirmed by polymerase chain reaction (PCR). The results of reverse transcriptase polymerase chain reaction (RT-PCR) revealed that foreign gene was expressed in transgenic plants. Western blot analysis using HA monoclonal antibody showed that HA protein of 65 kDa was accumulated in all of the transgenic plants.