披衣藻葉綠體表達外源蛋白系統之建立

Abstract

葉綠體基因組具有多拷貝數之特性，將目標基因以基因槍法 (particle bombardment) 送入葉綠體，經同源重組 (homologous recombination) 後可嵌入葉綠體基因組大量表達。本研究期望透過含篩選標誌基因 (selection marker gene) 或不含篩選標誌基因 (marker free) 兩種方法篩選擬轉殖系後，得到具有多拷貝數之轉殖基因組，以達到大量表現目標蛋白之目的。披衣藻 (Chlamydomonas reinhardtii) 生命週期較其他高等植物短，因此可以縮短篩選擬轉殖系的時間，同時披衣藻葉綠體亦具有可正確表現需正常摺疊之複雜構造蛋白，因此是作為生產外源蛋白之理想平台。將豬生殖與呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus, PRRSV) 之封套醣蛋白 (GP5)、膜蛋白 (GP6) 以及大腸桿菌忌熱性腸毒素B次單元 (heat-labile enterotoxin B subunit, LTB) 基因構築至轉殖載體中，以基因槍轉殖法 (biolistic bombardment) 轉殖至披衣藻葉綠體中。經反覆繼代與篩選進行均質化，以四組特異性引子對進行聚合酶連鎖反應分析，顯示目標基因已嵌入披衣藻葉綠體基因組中，惟尚未達均質狀態，且藍光下未見報導基因綠色螢光蛋白之表達。以反轉錄聚合酶連鎖反應分析，顯示目標基因確實進行轉錄作用。以LTB以及PRRSV專ㄧ性抗體進行西方免疫轉漬分析目標蛋白表達，結果顯示無目標蛋白累積之現象。因此本研究以影響葉綠體表達外源蛋白之因素作深入探討，期望以披衣藻作為大量表現外源蛋白之平台，以生產植物性口服疫苗。

Chloroplast genome occurs in high copy numbers. High levels transgene expression can be achieved by target gene integration to chloroplasts through site-specific homologous recombination. Presumably, chloroplasts also have the ability to express eukaryotic proteins with correct folding leading to biologically active proteins. The life cycle of Chlamydomonas reinhardtii is shorter than other higher plants. Therefore, C. reinhardtii provides an ideal platform for foreign protein production. In this study, the transplastomic lines were obtained after selection with or without marker. The envelope glycoprotein GP5 and non-glycoprotein GP6 from porcine reproductive and respiratory syndrome virus (PRRSV) were fused with heal-labile enterotoxin B subunit gene and transformed into chloroplast genome of C. reinhardtii by particle bombardment. Attempts were made for obtaining homoplasmic transplastomic cell lines after several rounds of subcultures. Polymerase chain reaction was carried out with four specific primer pairs to confirm the insertion of target genes. The transcription of target gene was then analyzed by reverse transcription polymerase chain reaction. Western blot analysis of target protein using anti-LTB monoclonal antibody and anti-PRRSV polyclonal antibody showed there were no target protein accumulated.