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標題: 以洛德乳酸桿菌疫苗載具pESV-nlp1表現腸毒型大腸桿菌之毒素融合蛋白STLTb,並評估其免疫效力
Evaluation of an expression-secretion vector of the product-cell wall-anchorage type, pESV-nlp1, for use in Lactobacillus reuteri as vaccine carrier
作者: 鄭憲覬
cheng, Hsien-Chi Hsieng-Chi
關鍵字: Lactobacillus reuteri
vaccine carrier
出版社: 獸醫微生物學研究所
摘要: 中文摘要 經由黏膜免疫法誘導產生保護性抗體的方式,已在近十年來的疫苗發展上逐漸受到重視,而在眾多引起黏膜免疫法中,應用於抗原攜帶體之方式,就以乳酸菌(lactic acid bacteria, LAB)被公認為是最安全且有效的攜帶體,主要原因除了他們屬於GRAS (generally regarded as safe)微生物對人及動物安全無虞,其本身還具有佐劑功能及低免疫原性之特點,且可在腸道黏膜中生長拓殖。因此,為使洛德乳酸桿菌(Lactobacillus reuteri)也可以應用於疫苗載體的開發,邱等(2002)已構築完成一個具有Lactococcus lactis的脂蛋白啟動子(lipoprotein promoter)之表現分泌載體pESV-nlp1;針對此一載體系統,我們利用綠螢光蛋白(green fluorescent protein, GFP)及阿爾發澱粉水解酶(-amylase)分別進行表現特性之分析,結果顯示所表現的異源性蛋白質會嵌合於洛德乳酸桿菌的細胞膜上。因此本實驗利用重組技術將兩種具有黏膜佐劑功能的蛋白基因,分別是鼠介白素6 (mouse interlukin 6)或熱不穩定毒素之a次單位蛋白突變型 (mutant from of heat-labile toxin a subunit/R192G, LTm),與腸毒型大腸桿菌(enterotoxigenic strain of Escherichia coli)之毒素融合蛋白(heat–stable toxin linked to heat-labile toxin b subunit, STLTb)基因連結在一起,經轉殖於pESV-nlp1表現分泌載體後,電孔轉形至洛德乳酸桿菌DSM20016及野鼠腸道分離菌株3-3中,以進行蛋白質之表現試驗,在細胞壁發現大部分的重組蛋白。本實驗進而評估在餵食洛德乳酸桿菌轉形株於小鼠後,是否可產生有效力價的中和性抗體及免疫的保護功能;結果顯示餵食攜帶pESV-nlp1衍生載體之洛德乳酸桿菌轉形株3-3可使小鼠產生較高的抗體力價,其中又以毒素融合蛋白與黏膜免疫佐劑LTm合併使用可產生較好的保護效力,並證實洛德乳酸桿菌表現載體pESV-nlp1可作為疫苗載具之開發與應用。
One of the new vaccine strategies, for recent decade years, has been emphasized on vaccination with mucosal induction way. Among numerous bacteria being used as the host for mucosal vaccine delivery system, LAB (lactic acid bacteria) have been found to be a good candidate for this purpose, since they are naturally equipped with several advantageous properties, including GRAS status, adjuvant properties, mucosal adhesive properties and low intrinsic immunogenicity. In an attempt to develop L. reuteri as a vaccine carrier, an efficient expression-secretion (ES) vector, pESV-nlp1, has been constructed by using a lipoprotein promoter from Lactococcus lactis to drive the transcriptional machinery for this cloning system. Further cloning of a green fluorescent protein (GFP) gene and a α-amylase gene as reporters into pNLP1 allowed us to verify this vector as a cell wall-anchorage type of ES system. We fused two mucosal adjuvant proteins, i.e., mouse interleukin 6 (mIL-6) and LTa mutant protein (LTa/R192G), respectively, to the toxin fusion protein STLTb from enterotroxigenic strain of Escherichia coli (ETEC) by genetic recombination and expression techniques. The resultant plasmids of pNLP1-STLTb derivatives were successfully transformed into Lacatobacillus reuteri DSM20016 and 3-3 strains and the heterologous fusion proteins were found to consistently express on the cell wall of L. reuteri. Subsequent evaluation of the immune response and protection efficiency in BALB/c mice, orally administered with our pNLP1-STLTb derivatives, has proved that Lactobacillus reuteri are truely potential for use as vaccine vehicle.
Appears in Collections:微生物暨公共衛生學研究所



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