利用葉綠體轉殖菸草生產豬生殖與呼吸綜合症 病毒蛋白之研究

Studies on the Production of Reproductive and Respiratory Syndrome Virus Protein by Transplastomic Tobacco

葉至軒(Chin-Shuan Yeh)

指導教授 ： 黃鵬林

共同指導教授 ： 杜宜殷(Yi-Yin Do)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10510

摘要

豬生殖與呼吸綜合症 (porcine reproductive and respiratory syndrome, PRRS) 為由PRRS 病毒 (PRRSV) 所引起的病毒性豬隻傳染病，其病癥為母豬生殖障礙及各年齡層豬呼吸道感染等，造成養豬業之嚴重經濟損失。利用葉綠體轉殖植物表達外源蛋白，因葉綠體基因套數可達一萬套，且無基因靜默及位置效應，相較核轉殖植物能預期大幅提高表現量，且其為母本遺傳，故可避免外源基因擴散至環境中，故為良好之外源蛋白生產平台。本研究為大量生產PRRSV之次單位疫苗，將融合可誘發宿主免疫反應之病毒封套醣蛋白 (GP5) 、膜蛋白 (M) 與忌熱性腸毒素B次單元 (heat-labile enterotoxin B subunit, LTB) 基因之構築，利用基因槍轉殖法 (biolistic bombardment)，轉殖至菸草及香蕉之葉綠體基因組中。經反覆切葉誘導芽體再生以促進均質性，擬轉殖菸草在藍光下可見報導基因綠色螢光蛋白之表達，以三組特異性引子對進行聚合酶連鎖反應分析，顯示目標基因嵌入菸草葉綠體基因組中，但尚未達均質狀態。再以反轉錄聚合酶連鎖反應證明轉殖株確實表現目標基因，並利用GP5專一性抗體進行西方免疫轉漬分析轉殖株葉片蛋白，可偵測到預期為56 kDa的目標蛋白，經酵素連結免疫吸附分析目標蛋白表達量約佔 3.6% 總可溶性蛋白 (total soluble protein)。而在擬轉殖香蕉胚性細胞中，可觀察到其報導基因綠色螢光蛋白之訊號隨篩選時間而增強，而以連鎖聚合酶反應分析，顯示目標基因成功插入香蕉基因體中。本研究之結果顯示葉綠體轉殖確實可提升目標蛋白表達量，具有作為 PRRS 口服疫苗表達平台之潛力。

關鍵字

植物性疫苗 ； 封套醣蛋白 ； 膜蛋白

並列摘要

Porcine reproductive and respiratory syndrome (PRRS) is a swine viral infectious diseases which is induced by PRRS virus (PRRSV). The symptoms of PRRS including reproductive failure and severe respiratory tract illness cause great economical loss of swine industry. The chloroplast transformation system has several advantages over nuclear transformation system in foreign protein production, such as higher expression level due to the high plastome copy number and lack of position effect and gene silencing. The maternal inheritance of chloroplast greatly lower the possibility of gene contamination with closed-relative plant species. These advantages make transplastomic plants as a good platform for foreign protein production. To produce large amount of vaccine subunit, the host immunological response induction genes, the envelope glycoprotein (GP5) and matrix protein (M) of PRRSV were fused with heat-labile enterotoxin B subunit gene and transformed into chloroplast genome of tobacco and banana by biolistic method. The fluorescence of green fluorescence protein was detected in putative transplastomic tobaccos. Polymerase chain reaction was carried out with three specific primer pairs to confirm the insertion of target genes. The transcription of target gene was then analyzed by reverse transcription polymerase chain reaction and showed target fragment. Western blot analysis of leaf protein using anti-GP5 monoclonal antibody showed the expected signal with size about 56 kDa. The protein expression level of antigen in transplastomic tobacco leaves was about 3.6% of total soluble protein determined by enzyme-linked immunosorbent assay. The intensity of report gene increased as time goes by in transplastomic embryogenic banana cells. The target gene was amplified using specific primer pair confirmed the insertion of target gene into banana chloroplast genome. These results indicated the potential of transplastomic tobacco as a production platform of PRRSV oral vaccine.

並列關鍵字

plant-based vaccines ； envelope glycoprotein ； matrix protein

參考文獻

王志昇、許信剛、童德文、邢福珊、陳曦、唐麒麟、寧蓬勃. 2010. PRRSV 陝西分離株 ORF5 基因克隆序列分析及原核表達. 西北農業學報 19:1-6.

牛登元、王一成、袁秀芳、徐麗華、劉鄭、伏小平. 2009. PRRSV 浙江株 ORF5基因的原核表達與免疫原性分析. 浙江農業學報 21:126-129.

田小輝、王愛萍、喬松林、張改平、肖治軍、馮麗麗、劉運超、李喬木、李卓陽. 2008. 豬繁殖與呼吸障礙綜合症病毒 ORF5 基因的克隆與表達. 河南農業科學 2:103-106.

江雲波、方六榮、肖少波、謝甜甜、陳煥春. 2005. 豬繁殖與呼吸綜合症病毒 (PRRSV) GP5-M 在大腸桿菌中的融合表達及其ELISA診斷方法的建立. 生物工程學報 21:259-264.

周宏專、徐福州、王金洛、楊新建、史愛華、楊兵. 2007. 豬繁殖與呼吸綜合症病毒 ORF5 基因不同編碼區的表達與反應源性鑑定. 華北農學報 22:12-15.