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目的用原代培养的猫肠上皮细胞(IECs)对已构建的弓形虫T7噬菌体展示文库进行筛选,期望得到将新的弓形虫入侵宿主细胞黏附相关蛋白基因。方法将猫IECs与混有噬菌体文库的DMEM培养基孵育培养后进行洗脱筛选,再与宿主菌BLT5403共同培养扩增文库,筛选噬菌体DNA并进行基因序列测定与分析。结果洗脱下的噬菌体滴度从第1轮7.6×105到第5轮的3.6×107,富集了230倍,非特异性结合和亲和力低的噬菌体被成功去除;核苷酸序列分析表明含插入片段为394bp的噬菌体富集量最大,经检索为弓形虫棒状体蛋白基因,是棒状体蛋白基因ROP2和ROP8的共有序列。结论初步证明弓形虫棒状体蛋白为黏附相关蛋白基因,其表达产物为ROP2。
Objective To screen the T7 phage display library of Toxoplasma gondii using primary cultured human intestinal epithelial cells (IECs), and to obtain the new Toxoplasma gondii invasion host cell adhesion related protein gene. Methods Incubate and incubate cat IECs and DMEM mixed with phage library for elution and screening. Then amplify the amplified library with host strain BLT5403, screen the phage DNA for gene sequencing and analysis. As a result, the phage titer eluted was successfully removed from the first round of 7.6 × 105 to the fifth round of 3.6 × 107, and the phage enriched by 230 times, non-specific binding and low affinity were successfully removed. Nucleotide sequence analysis showed that the inclusion- The fragment with 394bp contained the largest amount of phage. After retrieval of the Toxoplasma gondii rod-like body protein gene, it was a consensus sequence of ROP2 and ROP8. Conclusion Toxoplasma gondii prolactin is an adhesion-related protein and its expression product is ROP2.