段倩玉;蒋蔚;陈芸;张曼玉;曹敬政;王权;

• 1:中国农业科学院上海兽医研究所
• 2:南京农业大学动物医学院

摘要(Abstract)：

为了研究刚地弓形虫ROP18的生物学功能,并深入了解其在抵抗宿主天然免疫方面所发挥的作用,本研究进行了弓形虫RH株ROP18基因的表达与鉴定。采用PCR方法扩增ROP18的截短基因,将其克隆至原核表达载体pET-30a(+)中后,转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导表达并优化表达条件,SDS-PAGE分析表达情况,重组蛋白纯化后免疫小鼠制备多克隆抗体,通过Western blot及IFA鉴定重组蛋白的免疫活性。结果表明:以弓形虫RH株基因组DNA为模板,成功扩增出长约678 bp的ROP18截短基因,并构建了原核表达质粒pET-30a-ROP18,测序结果与GenBank参考序列比对,同源性为100%;SDS-PAGE结果显示,重组蛋白ROP18以包涵体形式存在,分子质量约为31 kDa,且在IPTG终浓度为0.2 mmol/L、37℃诱导表达6 h时,表达量最高;Western blot结果显示重组蛋白ROP18可被感染弓形虫的犬阳性血清识别,表明ROP18有良好的反应原性;IFA结果显示,ROP18主要分布于弓形虫速殖子的胞浆内,且棒状体常存在的部位呈现较强荧光。本研究成功获得重组蛋白ROP18及针对ROP18蛋白的多克隆抗体,为ROP18特性与功能的研究奠定基础。

关键词(KeyWords)： 弓形虫;ROP18;原核表达;多克隆抗体

Abstract:

Keywords:

基金项目(Foundation): 上海市科技兴农创新项目【沪农科创字(2019)第3-3号】;; 上海市自然科学基金(17ZR1437300)

作者(Author): 段倩玉;蒋蔚;陈芸;张曼玉;曹敬政;王权;

Email:

DOI:

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