日本血吸虫带电多泡体蛋白5的生物学特性研究  

Characterization and expression analysis of charged multivesicular body protein 5 from Schistosoma japonicum

作　　者：张旻[1] 傅志强[2] 林矫矫[2] 洪炀 ZHANG Min;FU Zhi-qiang;LIN Jiao-jiao;HONG Yang(College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,China;National Reference Laboratory for Animal Schistosomiasis,Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research),NHC Key Laboratory of Parasite and Vector Biology,Shanghai 200025,China)

机构地区：[1]河南科技大学动物科技学院,洛阳471023 [2]中国农业科学院上海兽医研究所,国家动物血吸虫病参考实验室,上海200241 [3]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心),国家卫生健康委员会寄生虫病原与媒介生物学重点实验室,上海200025

出　　处：《中国人兽共患病学报》2022年第9期764-770,共7页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.31702226,No.31872256);上海市自然科学基金(No.19ZR1468900)联合资助。

摘　　要：目的克隆、表达日本血吸虫(Schistosoma japonicum,S.japonicum)带电多泡体蛋白5(CHMP5),并对其生物学特性进行研究。方法利用PCR扩增S.japonicum CHMP5基因目的片段,构建重组质粒pET28a(+)-CHMP5,并对该基因进行生物信息学分析。利用荧光定量PCR检测CHMP5在不同时期虫体中的转录水平。对体外表达的重组CHMP5蛋白进行纯化与质谱鉴定,并通过Western blotting检测其反应原性。利用免疫组化实验分析CHMP5在虫体中的分布。以小鼠为动物模型,评估重组CHMP5蛋白抗日本血吸虫病的免疫保护效果。结果S.japonicum CHMP5的开放阅读框为675 bp,编码224个氨基酸,具有6个B细胞抗原表位;主要定位于S.japonicum虫体的体被上。CHMP5基因在35 d和42 d虫体中的转录水平高于其它检测时期,且42 d雌虫中的转录水平约是42 d雄虫的3倍。克隆表达的重组CHMP5蛋白其理论分子量约为29 kDa,具有较好的反应原性;用其免疫小鼠24 d,即可使小鼠产生较高水平的IgG抗体,并最终诱导产生18.96%的减虫率和42.98%的肝脏减卵率。结论成功克隆、表达了S.japonicum体被蛋白CHMP5,其在不同发育时期虫体中均有表达,获得的重组CHMP5蛋白可诱导小鼠产生部分的免疫保护效果,为深入探讨S.japonicum CHMP5的生物学功能提供了基础。The aim of this study was to investigate the biological characteristics of charged multivesicular body protein 5(CHMP5)from Schistosoma japonicum.S.japonicum CHMP5 was amplified by PCR and cloned into the expression plasmid pET28a(+),then analyzed in bioinformatics software.The transcriptional level of CHMP5 in worms at different stages was analyzed by real-time PCR.Recombinant CHMP5 protein was expressed and identified by mass spectrometry,and its reactogenicity was analyzed by Western blotting.The localization of CH MP5 in worms was analyzed by immunohistochemistry.The immunoprotective effect against schistosomiasis induced by recombinant CHMP5 protein was evaluated in mice.The open reading frame of the S.japonicum CHMP5 gene was 675 bp,and it encoded a protein of 224 amino acids containing six B cell antigenic epitopes.Transcription of CHMP5 was up-regulated in worms at 35 d and 42 d,with respect to the other detected stages.The transcriptional level in 42-day-old female worms was almost two times higher than that in male worms.The 29-kDa recombinant protein was successfully expressed and had good reactogenicity.CHMP5 was distributed mainly on the teguments of worms.ELISA tests indicated high specific IgG antibody titers of mice vaccinated with the recombinant CHMP5 protein 24 days post immunization,with respect to those in the adjuvant control group and PBS control group.Mice immunized with the recombinant CHMP5 protein showed a 18.96%lower worm burden(P<0.05)and a 42.98%lower liver egg count(P<0.01)than adjuvant control mice.Thus,S.japonicum tegument protein CHMP5 was successfully cloned and expressed in all detected schistosome stages.The recombinant CHMP5 protein induced partial immunoprotection against S.japonicum in BALB/c mice.This study lays a foundation for future research on the biological functions of S.japonicum CHMP5.

关 键 词：日本血吸虫 带电多泡体蛋白5 转录水平 组织定位 免疫保护 

分 类 号：R383.2[医药卫生—医学寄生虫学]