國產轉基因木瓜之外源病毒蛋白過敏原序列同源性比對及其致過敏性評估

Abstract

木瓜輪點病毒(Papaya ringspot virus; PRSV)和木瓜畸葉嵌紋病毒(Papaya leaf-distortion mosaic virus; PLDMV)是台灣木瓜生產最主要的限制因子，目前仍無法以傳統的化學藥劑防治。根據病原誘導抗病性(pathogen-derived resistance)理論及植物基因轉殖技術，中興大學已育成單抗PRSV轉基因木瓜及雙抗PRSV與PLDMV轉基因木瓜，其可以有效地預防病害。 目前一般消費大眾對於基因改造作物(Genetically Modified Organism；GMO)仍有食用安全性的疑慮，故本實驗以雙抗轉基因木瓜(TPY10-4)為材料，進行轉基因木瓜過敏原性評估，包括序列同源性比對分析以及病毒鞘蛋白分析。 首先針對轉基因木瓜品系(TPY10-4)設計專一性引子組(TPY16-F, TPY16-R)，由轉基因木瓜之葉子、生果及熟果中皆可成功擴增出目標基因片段 (1328 bp)，經定序確認後，再與過敏資料庫比對，確認外來基因所轉譯的蛋白質並非是致過敏蛋白質。然後利用酵素聯結免疫吸附分析法檢測轉基因木瓜是否有病毒基因產物蛋白質存在。 進一步利用模擬胃液及模擬腸液，針對轉基因木瓜進行消化試驗，結果顯示，轉基因木瓜粗蛋白經ELISA分析後，所得之ELISA unit均小於0.2，與非轉基因木瓜ELISA unit相似，且均較PLDMV-infected leaf和PRSV-infected leaf粗蛋白之ELISA unit為低。結果顯示，即使轉基因木瓜中含有很微量之輪點病毒蛋白和畸葉嵌紋病毒蛋白亦會經消化液分解而消失，且轉基因木瓜與非轉基因木瓜差異並不大。因病毒鞘蛋白表現所引起之過敏性機率極低，故食用轉基因木瓜並無食用安全性的問題。 市售木瓜檢測方面，利用(Pn-1F/Pn-1R)、(TPY-F/TPY-R)和(NA/NB)三組引子組分別對十種不同販售地點之新鮮木瓜進行轉基因檢測。結果顯示，十種樣品均無檢測出轉基因成分，表示轉基因木瓜的流通程度並不高。

Papaya ringspot virus (PRSV) and Papaya leaf-distortion mosaic virus (PLDMV) were the major limiting factor of papaya production in Taiwan, these diseases caused by viruses were still cannot be prevented and controlled by traditional chemical agent. Nevertheless, based on the concept of pathogen- derived resistance and the plant-transgenic method, the domestic transgenic papaya resistant to PRSV and double resistant to both PRSV and PLDMV lines were successfully developed by National Chung Hsing University (NCHU). In recent years, the general populace were filled with anxiety about edible safety of genetically modified organism (GMO). In this study, the domestic transgenic papaya double resistant to both PRSV and PLDMV line (TPY10-4) were used for the experimental materials to conduct the allergenicity assessment. The allergenicity assessment included both sequence homology analysis and virus coat protein detection in this study. First, TPY16-F/TPY16-R primers were designed to amplify the 1328 bp DNA sequence of PLDMV-PRSV coat protein from transgenic papaya line TPY10-4. The expected DNA fragments were successfully detected from transgenic papaya leaf, raw fruit and ripe fruit samples. The sequence homology analysis of transgenic proteins from papaya samples using AllergenOnline and Allermatch database demonstrated that all transgenic protein possess < 35% identical with known allergens in the database. The virus coat protein contents in transgenic papaya detected by ELISA assay show negative results, indicating no virus coat protein expression in transgenic papaya fruit. On the other hand, the digestibily of transgenic papaya were assessed with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). After digestion, the results of ELISA showed that ELISA unit of transgenic papaya was extremely low (<0.2), and there was no difference between transgenic and non- transgenic papaya. The results showed that even if transgenic papaya contained both the PRSV and PLDMV coat protein, they was digested with SGF and SIF. Therefore, we considered that the transgenic papaya could be regarded as safe. Ten samples of papaya were purchased from the different stores. The primers (Pn-1F/Pn-1R, TPY-F/TPY-R and NA/NB) were used to investigate the transgenic ingredient of papaya samples. The result showed that all of ten samples have no transgenic ingredient.