日本血吸虫主要虫卵抗原Sjp40在感染新西兰白兔肝脏中的表达与免疫定位

摘要

Objective To examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits. Methods New Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas. Results The level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas. Conclusion The transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.%目的：观察Sjp40在感染新西兰白兔肝脏沉积虫卵及其肉芽肿病变形成中的表达情况，并进行免疫定位。方法日本血吸虫尾蚴感染新西兰白兔，收集未感染组、29 dpi组和45 dpi组肝脏，Trizol法提取各组肝脏总RNA，以日本血吸虫甘油醛-3-磷酸脱氢酶（GAPDH）为内参，Taqman探针qRT-PCR检测Sjp40 mRNA的表达；提取各组肝脏总蛋白，以硫酸铵盐析法和Protein G免疫亲和柱层析法纯化的抗Sjp40-McAb 9G7和抗弓形虫tSAG1-McAb Y3A8（对照抗体），western blot检测Sjp40蛋白的表达；制备肝脏石蜡切片，HE染色观察肝脏虫卵肉芽肿的形成与发展，进一步以免疫荧光技术进行Sjp40在肝脏沉积虫卵及其肉芽肿组织中的定位。结果日本血吸虫感染兔呈急性肝肉芽肿病变期的45 dpi肝脏沉积虫卵中Sjp40 mRNA水平显著高于29 dpi尚未形成虫卵肉芽肿结节的肝脏沉积的未成熟虫卵（P<0.05），westernb blot证实了Sjp40蛋白在29 dpi和45 dpi兔肝脏中的表达。免疫荧光显示：Sjp40在29 dpi兔肝脏中定位于未成熟虫卵内，而45 dpi可见感染兔肝脏中沉积大量成簇内含毛蚴的成熟虫卵及其周围肉芽肿组织均有明显荧光。结论 Sjp40在日本血吸虫感染兔肝脏沉积虫卵中的转录水平随虫卵发育成熟而显著增加，在感染兔急性肉芽肿病变期（45dpi）呈现由虫卵向其周围肉芽肿组织扩散现象，提示该分子在血吸虫肉芽肿形成与发展过程中具有重要作用。