关键词: 日本血吸虫, 脱尾童虫, 表膜结合肽, 转染效率, 抗虫效果

Abstract: Objective   To study the efficiency of ZLW/pEGFP-C2 plasmid transfection into Schistosoma japonicum schistosomula and observe its in vitro effect of anti-schistosomula.   Methods   Recombinant plasmid ZLW/pEGFP-C2 was transfected into mechanically transformed schistosomula by immersion in 0.75% DMSO and high concentration of plasmid. Enhanced green fluorescent protein （EGFP） transfected cells were observed under inverted fluorescence microscope. At 48 hours after culture, total RNA and proteins from transfected schistosomula were extracted, and the presence of the transgenes （ZLW and EGFP） in schistosomula were confirmed by RT-PCR and Western blotting. At 24, 48, 72， and 96 hours after transfection, the schistosomula were counted by light microscope with methylene blue staining. pEGFP-C2 empty plasmid group and TBS group served as controls.  Results   The transfection rate was about 10%. The fluor-escence of ZLW/EGFP protein was mainly localized in the tegument of the worms, especially abundant around oral sucker and ventral sucker. The expected size of 259 bp fragment was successfully amplified by RT-PCR and confirmed by DNA sequencing. Western blotting analysis showed that ZLW/EGFP was expressed in schistosomula. No statistically significant difference was established for schistosomula mortality among ZLW/pEGFP-C2 group （14.0%， 48.8%）, pEGFP-C2 group （15.9%， 45.7%） and TBS group （16.9%， 50.3%） at 24 and 48 hours after transfection （P＞0.05）. At 72 hours after trans-fection the mortality rate of ZLW/pEGFP-C2 group （92.7%） was significantly higher than that of pEGFP-C2 group （73.2%） （P＜0.01）, and after 96 h the mortality in ZLW/pEGFP-C2 group increased to 100%.  Conclusion   ZLW/pEGFP-C2 plasmid has been introduced into juvenile S. japonicum by immersion in 0.75% DMSO and high concentration of plasmid, and was expressed in the parasite.

Key words: Schistosoma japonicum, Schistosomulum, Tegument binding peptide, Transfection efficiency, Schistosomicidal effect