作　　者： (刘烨）; (赵桂华）; (尹昆）; (王洪法）; (肖婷）; (刘功振）; (仲维霞）; (崔勇）;

机构地区： 济南大学山东省医学科学院医学与生命科学学院,山东省寄生虫病防治研究所,济宁272033 山东省济宁市第一人民医院医学检验科

出　　处： 《中国血吸虫病防治杂志》 2017年第4期464-467,474,共5页

摘　　要： 目的建立猫肠上皮细胞(Intestinal epithelial cells,IECs)培养体系,构建其酵母双杂交cDNA文库,为筛选弓形虫毒性因子在终末宿主的互作蛋白奠定基础。方法分别采用组织块法以及低浓度胶原蛋白酶Ⅺ和中性蛋白酶Ⅰ联合消化法进行猫IECs的原代培养,通过形态学观察及细胞免疫组织化学方法鉴定后提取mRNA,运用SMART技术合成cDNA第一链,以LD-PCR扩增获得双链cDNA(ds cDNA),通过同源重组方法在酵母菌株Y187中构建猫IECs的cDNA文库,计算文库的容量,以酵母菌液PCR检测插入片段的大小分布。结果两种培养方法效果表明酶联合消化法更为有效。本研究建立了连续培养猫IECs体系,培养出高纯度的猫IECs,用其构建的cDNA文库容量为1.1×10~6,滴度为2.8×10~9cfu/ml,插入片段大小在0.5~2.0 kb之间。结论培养的原代猫IECs构建的cDNA文库符合酵母双杂交筛选互作因子标准,这为筛选弓形虫终末宿主的毒性因子互作蛋白奠定了基础。 Objective To establish the primary cat intestinal epithelial cells（IECs）culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker？Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1＆#215; 106 independent clones. The titer was 2.8 ＆#215; 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

关 键 词： 弓形虫 肠上皮细胞 猫 原代培养 酵母双杂交 文库