日本血吸虫表皮生长因子受体基因的鉴定和功能研究  

Identification and functional study of the Schistosoma japonicum epidermal growth factor receptor gene

作　　者：相慢玉 李健[1] 罗芳 孙成松 朱炳宽 王吉鹏[1] 莫筱瑾[2] 张颋[2] 徐斌[2] 冯正[2] 胡薇[1,2] XIANG Man-Yu;LI Jian;LUO Fang;SUN Cheng-Song;ZHU Bing-Kuan;WANG Ji-Peng;Mo Xiao-Jin;ZHANG Ting;XU Bin;FENG Zheng;HU Wei(State Key Laboratory of Genetic Engineering,Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering,Ministry of Education Key Laboratory of Contemporary Anthropology,School of Life Science,Fudan University,Shanghai 200433,China;National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Key Laboratory of Parasite and Vector Biology,National Health Commission,WHO Collaborating Centre for Tropical Diseases,Joint Research Laboratory of Genetics and Ecology on Parasite-Host Interaction,Chinese Center for Disease Control and Prevention&Fudan University,China)

机构地区：[1]遗传工程国家重点实验室、生物多样性与生态工程教育部重点实验室、现代人类学教育部重点实验室、复旦大学生命科学学院,上海200433 [2]中国疾病预防控制中心寄生虫病预防控制所、国家卫生健康委员会寄生虫与媒介生物学重点实验室、WHO热带病合作中心、中国寄生虫病预防控制中心-复旦大学寄生虫-宿主遗传与生态研究联合实验室

出　　处：《中国血吸虫病防治杂志》2020年第2期123-131,共9页Chinese Journal of Schistosomiasis Control

基　　金：National Natural Science Foundation of China (31725025 and 81271867)。

摘　　要：目的鉴定日本血吸虫表皮生长因子受体基因(SjEGFR),并阐明其在日本血吸虫生长和生殖发育中的作用。方法通过5’-RACE和3’-RACE扩增、测序获取SjEGFR基因全长片段。采用荧光定量PCR(qPCR)技术检测该基因在日本血吸虫不同发育阶段(16、18、20、22、24、26 d)表达情况。使用整条虫体原位杂交法对感染24 d的日本血吸虫雌、雄虫SjEGFR基因转录本进行定位。通过体内持续RNA干扰(RNAi)技术特异性敲低SjEGFR基因,待虫体发育至30 d收集虫体。统计分析雌、雄虫回收数量以及虫体长度,通过明矾卡红染色观察虫体生长发育情况。结果首次鉴定了SjEGFR基因,其结构具有一个保守的酪氨酸激酶位点。整条虫体原位杂交定位显示该基因转录本在虫体各处均有表达,在雌、雄虫肠道部位呈现出明显着色的高表达。体内RNAi显示,SjEGFR基因表达被特异性敲低后,雌、雄虫生长受阻。RNAi组雌虫子宫内无虫卵,肠道内无色素沉积、卵黄腺发育迟滞、卵巢发育受阻;雄虫呈现睾丸个数减少、体积变小,出现了更多未成熟的精母细胞。结论特异性敲低SjEGFR基因表达可影响日本血吸虫生长和生殖发育,阻滞虫卵产生。Objective To characterize the epidermal growth factor receptor(EGFK)gene in Schistosoma japonicum(SjEGFR gene)and investigate the role of the EGFR gene in regulating the growth,reproductive system,maturation and fecundity of S.japonicum.Methods Rapid amplification of cDNA ends(RACE)was performed to obtain the full length of the SjEGFR gene,and the SjEGFR gene expression was quantified in different developmental stages of S.japonicum using a quantitative real-time PCR(qPCR)assay.The tissue localization of the SjEGFR gene was detected in 22-day parasite using whole-mount in situ hybridization(WISH).Following RNA interference(RNAi)-induced knockdown of the SjEGFR gene,the worm length,pairing rate and worm burden of S.japonicum were measured,and the worm morphology was observed using optical microscopy and confocal microscopy.Results The SjEGFR gene was identified with a conserved tyrosine-kinase active site,and the SjEGFR gene expression was detected at various developmental stages in male and female parasites.WISH showed that the transcript of the SjEGFR gene was localized on the tegument and in the digestive organs of S.japonicum.RNAi-induced SjEGFR knockdown resulted in marked suppression of the worm growth,smaller size of male testicles that contained more immature spermatocytes,and apparent impairment of ovary and vitelline gland development.In addition,no eggs were found in the uterus of SjEGFR knocked-down female parasites,indicating the interruption of egg production.Conclusions Inhibition of SjEGFR expression may remarkably suppress the growth and maturation of S.japonicum,and interrupt the egg production.

关 键 词：日本血吸虫 表皮生长因子受体 生殖 成熟 

分 类 号：R383.24[医药卫生—医学寄生虫学]