日本血吸虫童虫表膜结合多肽的亲和筛选与初步鉴定  

Screening and Characterization of Peptides Specifically Binding to the Schistosomulum Tegument of Schistosoma japonicum

作　　者：魏琦[1,2] 刘彦[2] 曾庆仁[2] 陈欲晓[1] 周军[3] 刘碧源[1,2] 邹艳[4] 杨胜辉[2] 蔡力汀[2] 李立新[1] 兰玲梅[2] 

机构地区：[1]中南大学基础医学院免疫学教研室,长沙410013 [2]中南大学基础医学院细胞与分子生物学实验中心,长沙410013 [3]中南大学湘雅三医院实验中心,长沙410013 [4]湖南中医药高等专科学校基础医学部,株洲410013

出　　处：《中国寄生虫学与寄生虫病杂志》2010年第3期172-175,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30570952);湖南省卫生厅(2009)项目~~

摘　　要：目的筛选噬菌体十二肽库中与日本血吸虫(Schistosoma japonicum)童虫表膜特异性结合的多肽并鉴定。方法利用噬菌体十二肽库与日本血吸虫活童虫靶分子之间的亲和力结合,经3轮吸附-洗脱-扩增,从末次回收的结合噬菌体中随机挑取20个克隆进行测序。根据测序结果 ,选取出现次数最多的噬菌体克隆作为目标噬菌体,免疫组化检测目的噬菌体,并回输到已感染日本血吸虫的小鼠体内,2.5h后处死小鼠,回收肝脏和日本血吸虫童虫,分别洗脱与肝脏和童虫结合的噬菌体,进行统计学分析。结果经3轮筛选后,噬菌体回收率从第1轮的0.77×10-8到第3轮的0.75×10-5,说明噬菌体得到了有效富集。DNA测序结果表明,20个噬菌体克隆中的15个克隆呈现QHPRIRKOOOOO序列。免疫组化结果显示,表达该序列的噬菌体能与童虫表膜有效结合;体内回输试验证实,该噬菌体能有效地靶向结合于体内日本血吸虫童虫表膜。结论筛选获得的短肽QHPRIRKOOOOO能有效地靶向结合日本血吸虫童虫表膜。Objective To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum tegument of Schistosoma japonicum. Methods A 12 phage-display peptide library was screened with the S. japonicum schistosomula as the target cells for biopanning by degrees, positive clones picked randomly were deduced by DNA sequencing. According the sequencing result, immunohistochemical staining was performed to determine the specificity of the phages to the tegument. To test their targeting efficacy, the interested phage clones were infused back to the mice infected with S. japonicum, mice were sacrificed 2.5 hours later, and the phage distribution in the liver and the tegument of schistosomula was appraised, respectively. Results After 3 rounds of biopanning, the phage recovery rate increased from 0.77×10-8 to 0.75×10^-5, indicating that the phage library was successfully enriched in the tegument of schistosomula. Seventy-five percent （15/20） of the analyzed sequences were identical with a sequence of QHPRIRKOOOOO. The immunohistochemical stainings showed this sequence specifically binding to the tegument. In vivo titering displayed that this sequence selectively targeted the tegument. Conclusion The peptide of QHPRIRKOOOOO specifically binds to the schistosomulum tegument.

关 键 词：日本血吸虫 童虫 噬菌体展示技术 表膜 靶向性 

分 类 号：R383.24[医药卫生—医学寄生虫学]