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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 identify peptides that specifically bind to Schistosoma japonicum schistosomula in phage docusin repertoire. Methods Using the binding affinity between the phage-dodecapeptide library and the target molecule of Schistosoma japonicum, 20 clones were randomly selected from the last recovered phage by three cycles of adsorption-elution-amplification. According to the sequencing results, the most phage clones were selected as the target bacteriophage. The target bacteriophages were detected by immunohistochemistry and transfused into mice that had been infected with Schistosoma japonicum. After 2.5 hours, the mice were sacrificed and the liver and Schistosoma japonicum were recovered. The phage that bound to liver and schistosomiasis were eluted separately for statistical analysis. Results After three rounds of screening, phage recovery rate from 0.77 × 10-8 in the first round to 0.75 × 10-5 in the third round showed that phage was effectively enriched. DNA sequencing results showed that 15 of the 20 phage clones presented the QHPRIRKOOOOO sequence. The results of immunohistochemistry showed that the phage expressing this sequence could bind effectively to Schistosoma japonicum surface membrane. The in vivo transfusion assay confirmed that the phage could effectively bind to the Schistosoma japonicum schistosomula surface membrane. Conclusion The short peptide QHPRIRKOOOOO obtained by screening can effectively target Schistosoma japonicum schistosomula surface membrane.