日本血吸虫Tsunagi蛋白基因的获得、表达及初步鉴定.pdfVIP

120 中国人兽共患病学报 2008. 24 (2) Chinese Journal of Zoonoses 文章编号: 1002-2694(2008)但-0120-05 日本血吸虫 Tsunagi 蛋白基囚的获得、表达及初步鉴定铸 王挠楠，雷黎，赵志荣，朱绍春，刘 蒜，沈际佳 摘 要:目的 获得日本血吸虫 Tsunagî 蛋白基园的 cDNA 序列，克盗、表达和纯化原核蛋白并初步鉴定。方法 应用简 并引物 PCR 和 5 端、3 端锚定 PCR. 扩增得到日本血吸虫 τsunagi 基因的完整齐放魏读框(Open reading frame. ORF). 将该 基因亚克隆入原核表达载体 pET28a{+) .诱导表达并纯化融合蛋白。用纯化后的蛋白免疫小鼠，制备该蛋白的多克隆抗体， 分别后 ELISA 和 Western Blotting 对表达蛋白进行初步鉴定。结果 获得日本血吸虫 Tsunagi 蛋白基囚的完整 ORF. 序列全 长为 531bp. 编码 177 个氨基酸，构建的重组质粒 pET28a-SjTsunagi 在大肠杆菌中获得了可洛性表达3 以重组蛋白免疫小鼠 获得效价为 1 51 200 的多克建抗体.Wcstern Blottíng 证实抗体可特异性识Jllj 目的蛋白 e 结论 成功获得日本血吸虫 Tsu­ nagi 蛋白编码基囚的全长。RF. 在大肠籽茵中克隆表达了 Tsu因gi 基因，并制备了特异性多克隆抗体.为进一步研究其在日本 血吸虫生殖生物学中的功能奠定了基地a 关键词:自本血吸虫 ;Tsunagi; 克隆;基因表达2 多克隆抗体 中理分类号;R383.24 文献标识码:Ä Cloning , expression and preliminary identification of tbe coding genetic sequences of the Tsunagi protein from Schistosoma j aponicum WANG Xiao-nan • LEI Li. ZHAO Zhi-rong , ZHU Shao-chun , LlU Miao , SHEN Ji-jia (Ðeþartment of Microbiology and Parasitology , Anhui Medical University. Hefei 230032. China) AßSTRACT: To obtaín the coding gcnetíc seq这ence of the Tsunagí protein from Schistosoma japonicum and to identify its antigcnicity. Dcgenerate PCR.5end and 3end anchored PCR were used to isolate the whole open reading frame (ORF) for this protein from S.jaþonícum cDNA library and adult cDNA. After confirmed by DNA sequencing. the encoding ORF was ampli- fíed and inserted into expression vector pET28以十) and thcn exprξssed as a fusion protein. The recombínant protein was p出i­ fied by an affinity column and specific antiserum was obtaincd from thc mice immunized with this protein. The titer and speci- ficity of the antiserum was determined by ELISA and Western blotting