以菸草細胞生產重組塵蟎過敏原蛋白Der p 2之研究

Abstract

重組過敏原蛋白已在許多不同系統中表現， 且植物系統被認為是具有競爭力的外源蛋白表現系統。本研究中，從已轉入CaMV 35S啟動子驅動塵蟎過敏原蛋白Dermatophagoides pteronyssinus 2 (Der p 2) 基因的轉殖煙草植株，來誘導癒傷組織與毛狀根，之後分別進行懸浮細胞培養與毛狀根培養，並利用自行建立之Der p 2三明治酵素免疫分析法(sandwich-ELISA)進行定量。從8株懸浮細胞株中挑選出Der p 2產量最高的細胞株S3，搖瓶培養16天，產量為788.3 μg/ L；以生物反應器進行批次培養，產量在第8天即達515.1 μg/ L。在轉植植株誘導毛狀根方面，共得24個細胞株，經30天三角搖瓶培養後，細胞株R19產量為475.2 ng/mL。此外，我們也建構以誘導性啟動子HSP 18.2融合Der p 2之嵌合基因，並在菸草毛狀根中獲得表現。在重組Der p 2蛋白的抽出與純化方面，重組蛋白粗抽液經硫酸銨沉澱、陽離子交換樹酯SP Sephadex、與自行建立之Der p 2單株抗體親合管柱處理後，可獲得以Der p 2為主要蛋白之溶液，收率為19.7 %。

Recombinant allergenic proteins have been produced in a variety of different expression systems. Plants and plant cells are now considered as viable and competitive expression systems for large-scale protein production. In this research, the plantlet of transgenic tobacco contained CaMV 35S promoter chimeric with Dermatophagoides pteronyssinus 2 (Der p 2) gene was used to induce formation of callus and hairy roots, and cultures of suspension cell and hairy root were studied for recombinant Der p 2 production. By Der p 2 sandwich enzyme-linked immunosorbent assay (sandwich-ELISA), cell line-D3 expressed the highest Der p 2 productivity in 8 suspension cell lines. The cultivation of S3 in flasks showed the maximum productivity, 788.3 μg/ L, at 16th d; batch culture of S3 in bioreactor reached the highest productivity, 515.1μg/L, at 8th d. Induction of hairy roots from transgenic tobacco, 24 cell lines was established. Maximum productivity, 475.2 ng/mL of Der p 2 was produced in flask within 30 d by R19. In this work, we constructed the chimeric gene of HSP 18.2 inducible promoter fused with Der p 2, and successfully expressed it in tobacco hairy roots by heat treatment. After the treatment of ammonia sulfate precipitation, cation exchange chromatography (SP Sephadex), and monoclonal anti-Der p 2 affinity column, recombinant Der p 2 protein was purified into homologous, the recovery rate was 19.7 %.