关键词: 日本血吸虫, 表膜, 噬菌体展示技术

Abstract: Objective  To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum, not cercaria, tegument of Schistosoma japonicum.  Methods  A 12 phage-display peptide library was screened with the S. japonicum schistosomula and cercariae as the target cells for biopanning by degrees, 15 positive clones were picked randomly and deduced by DNA sequencing. According the sequencing result, ELISA test, elution recovery test and immunohistochemical staining were performed to determine the specificity of the phages to the tegument. To further examine its binding properties, the positive peptide conjugated to RhB and recombinant pEGFP?-C2 plasmid were similarly synthesized.  Results  After 3 rounds of biopanning, the phage recovery rate increased from 3.50×10-5% to 3.20×10-2%, indicating that the phage library was successfully enriched in the tegument of schistosomula. The analyzed sequences were identical with 3 peptide sequence of ZL6, ZL4 and ZL1. ELISA showed that the P/N value of MppZL4, MppZL6 and MppZL binding the schistosomulum membrane protein was 6.72，3.65 and 2.22, while 1.58，5.15 and 1.20 of binding the membrane protein of cercariae, respectively. Elution recovery test showed that the elution recovery rate of MppZL4 ［（4.60±0.27）×10-2%］ was much higher than that of MppZL6 ［（2.10±0.23）×10-3%］, MppZL1 ［（1.20±0.28）×10-3%］ and M13KE［（1.30±0.60）×10-7%］（P<0.01）. Immunohistochemical staining showed that MppZL4 specifically bound to the tegument of schistosomula with a positive rate of 83.0%（83/100）. Fluorescent microscopy revealed that the synthesized RhB-ZL4 bound to the tegument of schistosomula. The ZL4/pEGFP-C2 plasmid was introduced into juvenile S. japonicum and expressed in the parasite.  Conclusion  The peptide of ZL4 specifically binds to the schistosomulum tegument but not to that of cercaria.

Key words: Schistosoma japonicum, Tegument, Phage display