利用轉殖菸草表達大腸桿菌忌熱性腸毒素蛋白之研究

Abstract

第二型豬環狀病毒(Porcine circovirus type 2, PCV2)是引起斷奶仔豬多系統衰弱綜合症(post-weaning multisystemic wasting syndrome, PMWS)的主要病原。已知PCV2之開放解讀框架(open reading frames, ORF)所產生的鞘蛋白(capsid)能誘發豬隻產生抗體；大腸桿菌忌熱性腸毒素B次單元(B subunit of heat-labile enterotoxin, LTB)是良好的抗原佐劑(adjuvant)；此外，組胺酸標誌(Histidine tag, His-tag)是一個能有效純化融合蛋白的親和性標誌；且內質網(endoplasmic reticulum, ER)保留訊息(retention signal)能提高融合蛋白的產量。故本研究將含有忌熱性腸毒素B次單元、linker序列、ORF2、His-tag及ER保留訊息序列不同組合之構築轉殖到菸草中，以轉殖菸草生產目標蛋白，期能作為口服疫苗。經GUS活性組織化學染色分析及PCR分析確認，目前已分別取得含有LTB、ER保留訊息及/或His-tag之融合蛋白基因的LTB2aE、LTB2bE、LTB2aHE、LTB2bHE、△2aHE及△2bHE的轉殖菸草。經西方轉漬分析(Western blot analysis)，不同構築轉殖株的融合蛋白表達量，只有LTB2aHE-5轉殖株具有目視可見的LTB片段，其表達量約為0.1% 總可溶性蛋白(total soluble protein, TSP)。南方氏雜交分析結果顯示，含有不同外源基因之轉殖株的拷貝數為1 ~ 3。拷貝數與西方轉漬分析結果比較，顯示外源基因之拷貝數與融合蛋白質表達量並無因果關係。

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). It is known that PCV2 ORF2 could serve as an antigen and B subunit of heat-labile enterotoxin (LTB) is a good antigen adjuvant. In addition, His-tag is an effective affinity tag for purification and the addition of an endoplasmic reticulum (ER) retention signal to a recombinant protein would stabilize its stability. In this study, production of fusion proteins of various combinations of LTB, linker sequence, ORF2, His-tag, and ER retention signal sequence in transgenic tobacco plants was conducted. Transgenic plants expressing one of the constructs of LTB2aE, LTB2bE, LTB2aHE, LTB2bHE, △2aHE, and △2bHE were obtained and the integration of target genes were confirmed by GUS staining and PCR analysis. The expression of target genes in these transgenic plants were demonstrated in fusion protein level by Western blot analysis. The only visible fragment in LTB protein levels of LTB2aE, LTB2bE, LTB2aHE and LTB2bHE transgenic plants is LTB2aHE-5 and the expression level is 0.1% TSP. Among all transgenic lines there are different copy numbers ranging from 1 to 3 based on Southern hybridization analysis in all transgenic lines. There is no correlation between copy number of forign genes and protein expression level in this study.