Development of live vaccine used transgenic Leishmania parasite

利什曼病通常是一种人畜共患病，其中狗是已知的最重要的动物宿主之一。为了控制这种人畜共患疾病，需要开发针对天然储库宿主的疫苗。在这项研究中，我们成功地产生，通过分子工程，利什曼原虫亚马逊前鞭毛体表达生物活性犬白细胞介素-8（cIL-8）。将cIL-8 cDNA的PCR产物插入到亚马逊乳杆菌表达载体（p6.5）中，该载体设计成含有用于选择的衣霉素（TM）抗性基因。将构建的表达载体p6.5-cIL-8电转化亚马逊乳杆菌克隆（C_1/12）的前鞭毛体。用TM培养基筛选转染细胞，用cIL-8特异性引物对转染细胞染色体外DNA进行PCR扩增，得到一个330 bp的预期产物。Western印迹分析表明，转染子的分子大小约为8.8kDa的肽，如预期的cIL-8。转染子培养上清对狗和小鼠多形核白细胞（PMN）具有趋化活性。

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).