【作者】 魏琦；

【导师】 陈欲晓； 曾庆仁；

【作者基本信息】 中南大学 ， 医学免疫学， 2010， 硕士

【摘要】 [目的]从噬菌体十二肽库中筛选出与日本血吸虫(Schistosoma japonicum, Sj)童虫表膜特异性结合的多肽,为研究血吸虫病的治疗提供靶向分子。[方法]利用噬菌体十二肽库与Sj活童虫靶分子之间的亲和力结合,经过三轮吸附-洗脱-扩增的筛选过程,从末次回收的结合噬菌体中随机挑取20个阳性克隆,首先对其进行测序；随后利用免疫组化、Western blot分析等方法观察目标噬菌体在体外与童虫表膜的结合能力,并通过动物实验将目标噬菌体回输到已感染血吸虫的动物体内,分别回收肝脏和血吸虫童虫,分别洗脱与肝脏和童虫结合的噬菌体,进行统计学分析,验证其靶向性。[结果]经3轮筛选后,噬菌体回收率从第1轮的0.77×10-8增加到第3轮的0.75×10-5,说明噬菌体得到了有效富集。DNA测序后发现,20个阳性噬菌体克隆中的15个克隆的插入序列均为QSFASLTNPRVL,将这些噬菌体克隆命名为ZW-15。免疫组化、Western blot均显示ZW-15噬菌体的该插入序列能与Sj童虫表膜有效结合。体内回输试验比较ZW-15及M13KE空载噬菌体的回收量,两者从单位重量虫体中的回收量差异显著(P1<0.01),而两者从单位重量肝脏中的回收量差别无统计学意义(P2>0.05),证实该ZW-15噬菌体的插入序列短肽能有效地靶向结合于体内Sj童虫表膜。[结论]本研究筛选获得阳性噬菌体克隆ZW-15所展示的短肽QSFASLTNPRVL能有效地靶向结合Sj童虫表膜,将为进一步研究血吸虫病的治疗提供靶向分子。更多还原

【Abstract】 [Objectives] To screen peptides specifically binding to the tegument of Schistosoma japonicum (Sj) schistosomula for potential use as targeting vectors for Sj therapy.[Methods] A 12 phage-display peptide library was screened with the Sj schistosomula as the target cells for biopanning by degrees, positive clones picked randomly were deduced by DNA sequencing. The immunohistochemical staining and Western blot were performed to determine the specificity of the phages to the tegument in vitro. To test their targeting efficacy, the interested phage clones were infused back to the mice challenged with Sj, and assessed by titering the phage distribution in both the livers and the tegument of schistosomula, respectively.[Results] After 3 rounds of biopanning, the phage recovery rates increased from 0.77×10-8 to 0.75×10-5, indicating that the phage library was successfully enriched in the tegument of Sj schistosomula. Seventy-five percent (15/20) of the analyzed positive phages were identical with a sequence of QSFASLTNPRVL, named as ZW-15. The immunohistochemical stainings and Western blot showed this sequence specifically binding to the tegument. Phage enrichment was examined by in vivo phage titerring. Compared with the control phage, Phage ZW-15 enrichment was significant in the tegument of schistosomula (P1<0.01), but not significant in the liver(P2>0.05), indicating the selective binding to the tegument by the peptide sequence.[Conclusion] The peptide of QSFASLTNPRVL specifically binds to the tegument of Sj schistosomula, which may have some significance for the future use as potential vectors in targeting therapy of Schistosomiasis.更多还原