Development of live vaccine used transgenic Leishmania parasite

使用转基因利什曼原虫寄生虫开发活疫苗

• 批准号: 08456149
• 负责人: MATSUMOTO Yoshitsugu
• 金额: $ 4.74万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456149/
• 关键词: IL-8; cDNA; expression vector; Leishmania; parasite; canine; 発現用ベクター; cDNA

Leishmaniasis is often a zoonosis in which dog is known to be one of the most important animal reservoirs. For control of such zoonotic deseases, development of a vaccine directed against the natural reservoir host is desirable. In this study, we successfully produced, by molecular engineering, Leishmania amazonensis promastigotes expressing biologically active canine interleukin-8 (cIL-8). The PCR products of cIL-8 cDNA were inserted into the L.amazonensis expression vector (p6.5) which was designed to contain a tunicanycin (TM) resistance gene for selection. The constructed expression vector (p6.5-cIL-8) was transfected into promastigotes of a L.amazonensis clone (C_1/12) by electroporation. Transfectants were selected in culture madium with TM.By PCR of extrachromosomal DNAs from the transfectants using cIL-8 specific primers, a predicted product of a 330 bp long was amplified. Western blot analysis of the trnsfectants demonstrated a peptide of about 8.8kDa in molecular size, as expected of cIL-8. The culture supernatant of the transfectant showed a chemotactic activity with dog and mouse polymorphonuclear cells (PMN).

利什曼病通常是一个人畜共患病，其中狗是最重要的动物储层之一。为了控制这种人畜共患病，希望开发针对天然储层宿主的疫苗。在这项研究中，我们通过分子工程成功地生产了亚马逊植物的利什曼原虫，表达生物活性犬白介素8（CIL-8）。将CIL-8 cDNA的PCR产物插入L.Amazonensis表达载体（P6.5）中，该载体旨在含有用于选择的外胞肌（TM）耐药基因。通过电穿孔将构造的表达载体（P6.5-CIL-8）转染到L.amazonensis克隆（C_1/12）的前差。使用CIL-8特异性引物从转染剂中选择转染剂，并用Tm.By PCR从转染剂中的tmby PCR进行选择，并扩增了330 bp的预测产物。 TRNS对染料的蛋白质印迹分析表明，正如CIL-8所预期的那样，分子大小的肽约为8.8kDa。转染剂的培养上清液显示狗和小鼠多形核细胞（PMN）的趋化活性。

项目成果

松本芳嗣ほか.: "Serological survey of the domestic dogs in Leishmaniasis-endemic dreas of Ecuador." Bulle Socie.Franco-Japonais Sci.Vet.7(1)(印刷中). (1997)

Yoshitsugu Matsumoto 等人：“厄瓜多尔利什曼病流行病中家犬的血清学调查。Bulle Socie.Franco-Japonais Sci.Vet.7(1)（出版中）”。

Mori M, Darco, C.A.et al.: "Seroepidemological aspects,Serological survey of the domestic doga in Ecuadcr." Bull.Soc.Franco-Japonais Sci.Vet.7(2). 30-41 (1997)

Mori M、Darco、C.A.等人：“血清流行病学方面，厄瓜多尔国内多加的血清学调查”。

Mori, M.Darco.C.A.at el.: "Seroepidemological aspects,Serologicalsurvey of the domestic doga in Ecuador" Bull.Soc.Franco-Japonais Sci.Vet.7(2). 30-41 (1997)

Mori, M.Darco.C.A.at el.：“厄瓜多尔国内犬的血清流行病学方面的血清学调查”Bull.Soc.Franco-Japonais Sci.Vet.7(2)。

Mori, M., Matsumoto, Y., et al.: "Seloepidemological, Serological survey of the domestic dog in Ecuador" Bull.Soc.Franco-Japonais Sci.Vet.7 (2). 30-41 (1997)

Mori, M.、Matsumoto, Y. 等人：“厄瓜多尔家犬的流行病学、血清学调查”Bull.Soc.Franco-Japonais Sci.Vet.7 (2)。