Stober Carmel B
Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, UK.
Methods Mol Biol. 2004;270:423-38. doi: 10.1385/1-59259-793-9:423.
A total of 2183 clones derived from four life cycle stage-specific, spliced-leader cDNA libraries of Leishmania major LV39 Neal strain were randomly picked and sequenced to generate expressed sequence tags (ESTs). Then 1094 unique genes were identified, with 18.2% having BLAST hits with known genes/proteins and 81.8% failing to match genes currently deposited in public databases. Approximately 250 unique genes were obtained from a lesion-derived amastigote complementary DNA (cDNA) library, the form of the parasite that is infective to the mammalian host. Polymerase chain reaction (PCR)-amplified ESTs were spotted onto glass slides, and DNA microarray used to identify a further approx 100 unique cDNAs highly expressed in amastigotes. One hundred unique, randomly selected amastigote-expressed genes were PCR amplified to exclude the 5'-spliced leader, and the full-length genes subcloned into the TOPO-TA cloning vector. The genes were sequence-verified, excised using restriction enzyme digestion, and cloned upstream of the eukaryotic cytomegalovirus promoter into the expression vector pcDNA3. Expression plasmids were sequence-verified and large-scale, endotoxin-free plasmids prepared. Then 100 microg of each expression plasmid (DNA vaccine) was delivered subcutaneously to the rump of susceptible BALB/c mice, the mice boosted 4 wk later and then challenged 2 wk post-boost with 2 x 10(6) L. major LV39 parasites to the hind footpad. Infection was monitored on a weekly basis by measuring footpad depth with digital calipers. Protection was scored by comparing the footpad depth of mice receiving empty vector DNA to those immunized with DNA containing L. major amastigote-expressed genes.
从利什曼原虫主要LV39尼尔菌株的四个生命周期阶段特异性、剪接前导cDNA文库中随机挑选了总共2183个克隆并进行测序，以生成表达序列标签（EST）。然后鉴定出1094个独特基因，其中18.2%与已知基因/蛋白质有BLAST匹配，81.8%与目前保存在公共数据库中的基因不匹配。从病变来源的无鞭毛体互补DNA（cDNA）文库中获得了约250个独特基因，无鞭毛体是对哺乳动物宿主具有感染性的寄生虫形式。聚合酶链反应（PCR）扩增的EST被点样到载玻片上，并用DNA微阵列鉴定出另外约100个在无鞭毛体中高表达的独特cDNA。对100个随机选择的无鞭毛体表达基因进行PCR扩增以去除5'-剪接前导序列，并将全长基因亚克隆到TOPO-TA克隆载体中。对基因进行序列验证，使用限制性酶切消化切下，然后克隆到真核巨细胞病毒启动子上游的表达载体pcDNA3中。对表达质粒进行序列验证并制备大规模无内毒素质粒。然后将100μg每种表达质粒（DNA疫苗）皮下注射到易感BALB/c小鼠的臀部，4周后对小鼠进行加强免疫，然后在加强免疫后2周用2×10⁶个利什曼原虫主要LV39寄生虫攻击后足垫。每周用数字卡尺测量足垫深度来监测感染情况。通过比较接受空载体DNA的小鼠与用含有利什曼原虫无鞭毛体表达基因的DNA免疫的小鼠的足垫深度来评分保护情况。
