蕃茄生產腸病毒71型VP1蛋白質之研究

A Study on the Production of EV71 VP1 Recombinant Protein in Tomato Plants

陳慧軒(Hui-Hsuan Chen)

指導教授 ： 王玉麒

國立臺灣師範大學/理學院/生物學系/碩士(2002年)

摘要

腸病毒71型（enterovirus 71, EV71）為一新興的惡性神經性腸內病毒，目前疫苗的開發研究刻不容緩。VP1是EV71的最主要外鞘蛋白，根據前人研究該蛋白質為生產次單元體疫苗的良好標的。本研究之目的即以VP1的基因為標的，建構基因表現卡匣，並轉形於蕃茄細胞之中，期望能產生含腸病毒食用疫苗的蕃茄果實。
本研究在建構基因表現卡匣時，共測試CaMV 35S啟動子及E8啟動子二種調控序列，在以農桿菌感染法及粒子槍法將基因表現卡匣轉形至蕃茄細胞後，乃在含hygromycin的培養基中進行篩選與植株分化的步驟。截至目前為止，共獲得含CaMV 35S啟動子的轉殖株20株及含E8啟動子的轉殖株18株。這些蕃茄轉殖株經以PCR方式檢測，確定均具有VP1基因的插入，而南方轉漬分析的結果則顯示各轉殖株中僅含有1~2個VP1基因的插入片段，以PCR檢測轉殖株的T1子代則確定VP1基因已穩定插入轉殖株的基因體內，並可遺傳給下一代。
蕃茄轉殖株內VP1基因表現的情形已進行RT-PCR及北方轉漬分析，目前在10株35S-VP1轉殖株及4株E8-VP1轉殖株的果實中測得有VP1 mRNA的表現；但西方轉漬分析目前尚無法偵測到VP1蛋白質的生成。由於上述RNA表現的結果顯示轉殖株內發生轉錄階層及轉錄後階層基因靜默情形的機會不高，我們推測無法測得VP1蛋白質的原因可能是由於VP1基因序列中含有蕃茄植株較少使用的密碼種類，以致造成轉譯效率降低。

關鍵字

蕃茄 ； 腸病毒71型 ； 食用疫苗

並列摘要

Enterovirus 71 (EV71) is a new neurotrophic virus that causes seasonal morbidity and mortality in children throughout the world with increasing frequency in recent years. Development of effective vaccines is the best choice among all control measures. VP1, the dominant surface protein of EV71, was reported to be a good candidate for making subunit vaccines. In this study, we tried to establish a fruit-based edible vaccine against EV71 by expressing recombinant VP1 protein in transgenic tomato plants.
To construct gene expression cassette containing VP1 gene, two different regulatory sequences of the CaMV 35S promoter and the E8 promoter were employed, respectively. After introducing the gene expression cassettes into tomato cells by Agrobacterium-mediated transformation or particle gun bombardment, transgenic plants were selected and regenerated in culture media with hygromycin. Presently, a total of 38 transgenic plants, including 20 with CaMV 35S promoter and 18 with E8 promoter, have been obtained. The results of PCR analysis revealed that all 38 transgenic plants contain both the VP1 and the hygromycine-resistance genes, while that of Southern blotting showed the insertion number of transgene was 1 to 2. Examination of T1 transgenic plants by PCR analysis confirmed the VP1 gene was stably and heritably inserted into the genome of these transgenic tomatoes.
The expression of VP1 transcript has been assessed by RT-PCR and Northern blot analyses. Our data identified the presence of VP1 transcript in 10 transgenic plants containing CaMV 35S promoter and 4 transgenic plants containing E8 promoter. However, the VP1 recombinant protein was currently undetectable in our Western analysis of transgenic plants. Since our data minimize the probability of transcriptional and post-transcriptional gene silencing mechanisms, the low abundance of VP1 recombinant protein may be due to the presence in the VP1 gene the codons rarely used by plants and thus resulting in low efficiency of protein translation.

並列關鍵字

tomato ； enterovirus 71 ； edible vaccine

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