采用弓形虫T7噬菌体文库筛选黏附相关蛋白基因的研究  

Screening of Toxoplasma adhesive protein genes from phage library

作　　者：崔勇[1,2] 仲维霞[1] 赵桂华[1] 李瑾[1] 徐超[1] 魏艳彬[1] 魏冬冬[1] 王洪法[1] 

机构地区：[1]山东省医学科学院、山东省寄牛虫病防治研究所,山东济宁272033 [2]济南大学,山东济南250022

出　　处：《中国病原生物学杂志》2013年第11期1005-1007,共3页Journal of Pathogen Biology

基　　金：山东省自然科学基金项目(No.ZR2010HQ062)

摘　　要：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 On the basis of T7 phage display library of Tozoplasma gondii was constructed the primary cul- tured IECs of cats to he used in order to screening thelibrary, expect to get a new T. gondii adhesion protein gene. Me- htods Firstly, IECs of cat mixed with DMEM medium contenting phage library was screened after incubate by eiution, then the library was amplified though common culture with host bacteria BLT5403 DNA genes which were screened. Results ted phage titer was increased at last sequencing and analysis phage rom 7.6 ×l0-5（first round） to 3.6 × 10-7 （five round）, which was enriched 230 times and successfully in addition to the low affinity and non-specific binding phage. Nucleotide sequence analysis showed that the most enrichment of phage is containing insert fragment of 394 bp, the insert fragment turned out to be T. gondii rod-like protein gene, which is the consensus sequence of the rod-like pro- tein gene 2（rop2） and 8（rop8）. Conclusion Preliminarily verify that the key protein for T. gondii and IECs interaction is T. gondii rhoptry protein. T. gondii rhoptry protein and cat intestinal epithelial cell specific binding whether or not remains to be further verified.

关 键 词：弓形虫 猫肠上皮细胞 噬菌体 黏附蛋白 棒状体蛋白 

分 类 号：R382.5[医药卫生—医学寄生虫学]