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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 a culture system of Intestinal epithelial cells (IECs) and construct a yeast two-hybrid cDNA library for the screening of Toxoplasma gondii toxic factor in the terminal host interaction protein. Methods The primary culture of feline IECs was carried out by tissue block method and digestion with low concentration of collagenase Ⅺ and neutral protease Ⅰ respectively. MRNA was identified by morphological observation and cellular immunohistochemistry, and then cDNA was synthesized by SMART technique Double strand cDNA (ds cDNA) was amplified by LD-PCR. The cDNA library of cat IECs was constructed by homologous recombination in yeast strain Y187. The capacity of the library was calculated. The size distribution of the inserted fragments was detected by yeast liquid PCR . Results The results of the two culture methods showed that enzyme digestion was more effective. In this study, a continuous incubation system of IECs was established to produce high purity cats IECs. The cDNA library constructed by this method had a capacity of 1.1 × 10 ~ 6, a titer of 2.8 × 10 ~ 9cfu / ml and an insert size of 0.5 ~ 2.0 between kb Conclusion The cDNA library constructed by primary cat IECs accorded with the standard of yeast two-hybrid screening interaction factor, which lays the foundation for the screening of Toxoplasma gondii Toxoplasma gondii interacting proteins.