日本血吸虫脱尾童虫表膜结合短肽的筛选与鉴定

发布时间：2018-05-28 14:51

本文选题：日本血吸虫 + 表膜　；参考：《中国寄生虫学与寄生虫病杂志》2013年01期

【摘要】：目的筛选噬菌体十二肽库中与日本血吸虫(Shistosoma japonicum)脱尾童虫表膜特异性结合而不与尾蚴表膜结合的短肽并鉴定。方法利用M13噬菌体十二肽库,经体外逆向差异筛选日本血吸虫尾蚴和脱尾活童虫,从第3轮回收的结合噬菌体中随机挑取15个克隆进行测序获取目标噬菌体后,采用ELISA法、洗脱回收率实验(以M13KE为阴性对照)和免疫组织化学法检测日本血吸虫脱尾童虫、尾蚴与噬菌斑克隆的特异性结合。荧光显微镜观察人工合成的阳性噬菌体短肽与日本血吸虫脱尾童虫体外的特异性结合。构建目的片段pEGFP-C2质粒体外转染日本血吸虫脱尾童虫。结果经3轮逆向差异筛选后.噬菌体回收率从第1轮的3.50×10~(-5)%到第3轮的3.20×10~(-2)%,富集度明显提高。DNA测序结果表明,15个噬菌体克隆分别含有ZL6、ZL4和ZL1等3个不同的短肽序列。ELISA结果显示,M13噬菌体短肽ZL4(MppZL4)、MppZL6和MppZL1分别与脱尾童虫膜蛋白结合后的P/N值为6.72,3.65和2.22,分别与尾蚴膜蛋白结合后的P/N值为1.58,5.15和1.20。洗脱回收率实验结果显示.MppZL4与脱尾童虫洗脱回收率[(4.60±0.27)×10~(-2)%]远高于MppZL6[(2.10±0.23)×10~(-3)%]、MppZL1[(1.20±0.28)×10~(-3)%]和M13KE[(1.30±0.60)×10~(-7)%)(P0.01)。免疫组化结果显示,日本血吸虫脱尾童虫与MppZL4特异性结合.阳性率为83.0%(83/100)。荧光显微镜检测结果显示,人工合成的RhB-ZL4可与日本血吸虫脱尾童虫体外特异性结合。构建的ZL4/pEGFP-C2质粒体外可成功转染日本血吸虫脱尾童虫。结论筛选获得的短肽ZL4能与日本血吸虫童虫表膜特异性结合.不与尾蚴表膜结合。
[Abstract]:Objective to screen and identify short peptides from phage dodeceptide library that bind specifically to the surface membrane of Schistosoma japonicum but not to the surface membrane of cercariae. Methods the M13 phage dodecapeptide library was used to screen Schistosoma japonicum cercariae and live schistosomiasis japonicum by reverse differential screening in vitro. 15 clones were randomly selected from the third cycle of the combined phage and sequenced to obtain the target phage. The phage was obtained by ELISA method. The specific binding of Schistosoma japonicum, cercariae and plaque clone of Schistosoma japonicum was detected by elution recovery test (using M13KE as negative control) and immunohistochemical method. The specific binding of synthetic phage short peptide to Schistosoma japonicum in vitro was observed by fluorescence microscope. The target pEGFP-C2 plasmid was constructed and transfected into Schistosoma japonicum in vitro. Results after three rounds of reverse differential screening. The phage recovery rate ranged from 3.50 脳 10 ~ (-5)% in the first round to 3.20 脳 10 ~ (-2) in the third round. The results of DNA sequencing showed that the 15 phage clones contained three different short peptides, ZL6, ZL4 and ZL1, respectively. Elisa results showed that the phage short peptides ZL4, MppZL4, MppZL6 and MppZL1. The P / N values after binding to the membrane proteins were 6.72 3.65 and 2.22, respectively, and the P / N values were 1.585.15 and 1.20 after binding to the membrane proteins of cercariae, respectively. The results of elution recovery test showed that the recoveries of 路MppZL4 and Thunb were significantly higher than those of MppZL6 [2.10 卤0.23) 脳 10 ~ (-3)%] and M13KE [1.30 卤0.60) 脳 10 ~ (-1) ~ (-3)%] and M13KE [1.30 卤0.60) 脳 10 ~ (-10) ~ (-7) ~ (-1) ~ (-1). Immunohistochemical results showed that Schistosoma japonicum was specifically bound to MppZL4. The positive rate was 83.0 / 100%. The results of fluorescence microscopy showed that the synthetic RhB-ZL4 could specifically bind to Schistosoma japonicum in vitro. The constructed ZL4/pEGFP-C2 plasmid was successfully transfected into Schistosoma japonicum in vitro. Conclusion the short peptide ZL4 can specifically bind to the surface membrane of Schistosoma japonicum. Not bound to cercariae surface membrane.
【作者单位】：南华大学医学院寄生虫学教研室;中南大学基础医学院细胞与分子生物学实验中心;
【基金】：教育部博士点基金博导类资助项目(No.20110162110029) 国家自然科学基金(No.81101274) 湖南省自然科学基金(No.10JJ3010) 湖南省教育厅优秀青年项目(No.11B106)~~
【分类号】：R392

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