利用植物表現豬生殖與呼吸道綜合症病毒疫苗基因之研究

Abstract

豬生殖與呼吸道綜合症病毒 (porcine reproductive and respiratory syndrome virus, PRRSV) 近年來嚴重感染豬群，造成養豬業重大經濟損失。以植物為基礎開發此病毒之多價次單位疫苗，為兼具經濟與方便之口服投與方式免疫的途徑。本研究將PRRSV ORF5基因分別以CaMV 35S啟動子及香蕉泛素基因啟動子啟動基因表現，並於抗原基因序列末端接上內質網保留信息及基質結合區序列，構築為疫苗表現載體，以農桿菌媒介法轉殖入香蕉及菸草。轉殖香蕉及菸草植株經抽取基因組DNA，以ORF5基因之專一性引子進行聚合酶連鎖反應(polymerase chain reaction)，可合成預期長度之片段，基因組南方氏雜交分析亦有預期之訊號片段，證實轉殖基因已確實插入植株基因組內。而以相對應於PRRSV之專一性抗體進行免疫轉漬分析可偵測到ORF5基因產物GP5蛋白之表達，經酵素連結免疫吸附分析(enzyme-linked immunosorbent assay)進行外源蛋白定量，轉殖菸草所表達之GP5蛋白量為112 ng/mg總水溶性蛋白(total soluble protein, TSP)，佔總水溶性蛋白0.01%；轉殖香蕉所表達的抗原量則為285 ng/mg TSP，約佔總水溶性蛋白0.03%。單獨表現ORF5基因之轉殖香蕉與菸草葉片經餵食豬隻後，可於豬隻血清及唾液內偵測到相對應之IgG及IgA產生。為進一步提高抗體效價及病毒中和能力，另進行修整及強化疫苗表現載體之構築，包括ORF5基因與LT-B基因融合，及ORF5與ORF6基因融合以形成異型雙元體等表現載體，經轉殖至香蕉及菸草，並以PCR、反轉錄聚合酶連鎖反應及南方氏雜交分析，進行轉殖株分子驗證，均可得到預期片段，而轉殖菸草所表達的LTB-GP5蛋白，均具有與GM1 ganglioside受體結合的能力。為提高香蕉轉殖成功率，使用不同處理方式搭配農桿菌媒介法進行轉殖，其中於農桿菌共培養過程中添加0.02% Pluronic F-68，具有較佳效果。為進一步提高抗原於植物中之表達量，構築香蕉及菸草葉綠體基因轉殖表現載體，經基因槍法轉殖入香蕉懸浮細胞及菸草葉片後進行抗生素篩選，香蕉則可見表達GFP綠色螢光之擬胚，而菸草轉殖株葉片及根部均可觀測到GFP報導基因的表現。

Porcine reproductive and respiratory syndrome (PRRS), also known as Blue-Ear Pig Disease, is an economically important disease induced by PRRS virus (PRRSV) in swine, which causes late-term reproductive failure and severe pneumonia in neonatal pigs. Molecular farming has a great potential since the cost for production is low and plant tissue is easy for use as food additive. The gene sequences of ORF5 corresponding to the neutralizing epitopes of PRRSV were constructed in the plant expression vector and expressed as subunit vaccines in banana and tobacco. Expected sizes of fragments were detected by polymerase chain reaction (PCR) and Southern hybridization, indicating that the ORF5 gene was integrated into the chromosomal DNA of the transgenic plants. Immunoblot analysis of proteins extracted from transgenic banana and tobacco plants were detected by PRRSV GP5 specific antibody, anti-PRRSV rabbit serum, and confirmed the expression of foreign gene. Quantification by enzyme-linked immunosorbent assay (ELISA) showed that GP5 protein levels reached 0.012% of total soluble protein in tobacco leaf samples, and 285 ng/mg, about 0.03% of total soluble protein in transgenic banana plants. Pigs fed with transgenic banana and tobacco leaves had developed immunity and showed a specific anti-GP5 response, demonstrating that GP5 proteins were expressed in plants and were able to induce the production of anti-GP5 antibodies in the pigs. To enhance the expression level and immunity of oral vaccine, ORF5 was fused with L-TB gene from E. coli. and ORF6 genes from PRRSV for expression in plants. The transformation event was validated by PCR, Southern analysis and reverse transcription polymerase chain reaction (RT-PCR). Plant-derived LTB-GP5 recombinant protein showed binding affinity to GM1 ganglioside receptor. To improve the efficiency of banana transformation system, different additives were evaluated during Agrobacterium- mediated transformation. Addition of 0.02% Pluronic F-68 while co-cultivation with Agrobacterium resulted in higher amount of GFP-expressed embryos than other treatments absent of Pluronic F-68. Another approach for enrichment of the target protein expressed in transgenic plants is through plastid transformation. Plastid transformation vectors specific for banana and tobacco were constructed, and transformed into banana and tobacco via particle bombardment. The GFP fluorescence was detected in transgenic banana embryos and tobacco leaves and roots.