日本血吸虫肝期童虫表膜结合多肽的筛选与初步鉴定

发布时间：2018-05-25 17:05

  本文选题：日本血吸虫 + 童虫　； 参考：《中南大学》2010年硕士论文

【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(P10.01),而两者从单位重量肝脏中的回收量差别无统计学意义(P20.05),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。
[Abstract]:[objective] to screen the peptides specifically binding to Schistosoma japonicum (SjjS) surface membrane from phage dodeceptide library, and to provide targeted molecules for the treatment of schistosomiasis. [methods] by using the affinity binding between phage dodeceptide library and Sj live child worm target molecule, 20 positive clones were randomly selected from the last recovered phage after three rounds of screening process of adsorption, elution and amplification. Then the binding ability of the target phage to the surface membrane of the child worm in vitro was observed by immunohistochemistry and Western blot analysis, and the target phage was transferred back to the infected animal by animal experiment. The liver and Schistosoma japonicum were recovered, the phage which was bound to the liver and the child worm was eluted, and the targeting of the bacteriophage was verified by statistical analysis. [results] after three rounds of screening, The phage recovery increased from 0.77 脳 10 ~ (-8) in the first round to 0.75 脳 10 ~ (-5) in the third round. The results showed that 15 of the 20 positive phage clones were inserted into QSFASLTNPRVL. the phage clones were named ZW-15. Immunohistochemical analysis showed that the insertion sequence of ZW-15 phage could effectively bind to the surface membrane of Sj child worm. The recoveries of ZW-15 and M13KE were compared in vivo. There was a significant difference in the recovery amount between the two species (P 10.01), but there was no significant difference in the recovery amount between the two species from the liver (P 20.05). It was confirmed that the inserted short peptide of the ZW-15 phage could be effectively targeted to the surface membrane of Sj children's worm in vivo. [conclusion] in this study, the short peptide QSFASLTNPRVL displayed by the positive phage clone ZW-15 can be effectively targeted to the surface membrane of Sj children's worm, which will provide a target molecule for further study on the treatment of schistosomiasis.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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中国期刊全文数据库 前10条

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