日本血吸虫ELAV-like 2基因的克隆、表达及功能分析

doi:10.11843/j.issn.0366-6964.2018.09.020

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (9): 1987-1997.doi: 10.11843/j.issn.0366-6964.2018.09.020

日本血吸虫ELAV-like 2基因的克隆、表达及功能分析

张媛媛^1,2, 许蓉^1,2, 何川川², 李肖纯^1,3, 程贵凤^1,2, 郭露², 刘金明², 李浩², 古少鹏^1*, 金亚美^2*

1. 山西农业大学动物科技学院, 太谷 030801;
2. 中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室, 上海 200241;
3. 上海师范大学生命与环境科学学院, 上海 200233

收稿日期:2018-03-06 出版日期:2018-09-23 发布日期:2018-09-23
通讯作者: 古少鹏,教授,硕士,主要从事兽医公共卫生与人畜共患病防治研究,E-mail:shpgu@163.com;金亚美,研究员,博士,主要从事寄生虫分子生物学与免疫学研究,E-mail:yameijin@shvri.ac.cn
作者简介:张媛媛(1989-),女,河北沧州人,硕士生,主要从事兽医公共卫生与人畜共患病防治研究,E-mail:2359866315@qq.com
基金资助:
国家自然科学基金（31672245）；上海市自然科学基金（16ZR1444100）

Cloning,Expression and Functional Analysis of ELAV-like 2 Gene of Schistosoma japonicum

ZHANG Yuan-yuan^1,2, XU Rong^1,2, HE Chuan-chuan², LI Xiao-chun^1,3, CHENG Gui-feng^1,2, GUO Lu², LIU Jin-ming², LI Hao², GU Shao-peng^1*, JIN Ya-mei^2*

1. College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China;
2. Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
3. College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200233, China

Received:2018-03-06 Online:2018-09-23 Published:2018-09-23

摘要/Abstract

摘要：

日本血吸虫成熟雌虫所产虫卵是造成宿主病理损害及疾病再传播的根源，因此从控制雌虫生殖发育入手对控制血吸虫病的发生具有重要意义。本研究通过5'-RACE和3'-RACE获得了在发育阻遏雌虫中高表达的SjELAV-like 2基因的全长序列，并对其进行克隆和表达，制备了相应的多克隆抗体，Western blotting分析结果表明该蛋白质具有良好的反应原性；实时定量分析发现该基因在血吸虫童虫时期高表达，成熟后趋于稳定，且在雌虫体内的表达量显著高于同时期合抱雄虫；该基因在雄虫体内的表达量基本恒定；免疫组化研究发现，在血吸虫成虫雌虫该蛋白主要定位于虫体体被。利用RNA干扰技术对该基因的生物学功能进行了初步研究，RT-qPCR结果显示，RNAi可明显抑制SjELAV-like 2基因的表达；对该基因表达的长期干扰导致受干扰虫体产卵能力及卵胚孵化能力的下降，统计分析发现，干扰组宿主每克肝荷卵减少达39.67%（P<0.01），虫卵的卵胚孵化减少达75.56%（P<0.01）。扫描电镜观察发现，与NC组相比，siRNA干扰组虫体体被上泡状突出物明显增多，这些结果表明SjELAV-like 2基因对日本血吸虫生长发育具有重要影响。

Abstract:

Schistosomiasis is a globally important helminthic disease of humans and animals,eggs produced by the mature female worms are the main factor that cause pathological damage and disease transmission. The study on controlling female reproductive development has great significance in controlling schistosomiasis. In this study, the full-length of SjELAV-like 2 gene sequence was attained by 5'-RACE and 3'-RACE. The SjELAV-like 2 coding sequence was successfully cloned and expressed, and the polyclonal antibody was produced. Western blotting analysis showed that the protein has good reactionogenicity. RT-qPCR analysis revealed that the gene had the highest expression in schistosomula and tended to be stable after maturation. The expression level of SjELAV-like 2 in the female was significantly higher than that in the male. Its expression in each periods of the male remained maintenance level. Immunohistochemical results indicated that the protein was mainly located in the tegument of Schistosoma japonicum in adult females. RNAi interference technology was employed to study the biological function of SjELAV-like 2 gene. RT-qPCR results showed that RNAi could effectively reduce the expression of SjELAV-like 2 gene in the parasites. The long-term RNAi also reduced the hatching rates about 75.56% (P<0.01), and reduced the liver egg burden about 39.67% (P<0.01). The scanning electron microscope showsed that there were more vesicular protrusions on the tegumental tissues of siRNA interference group worms compared with the negative control. These observations implied that SjELAV-like 2 has important influence on growth and development of Schistosoma japonicum.

中图分类号:

S852.735

张媛媛, 许蓉, 何川川, 李肖纯, 程贵凤, 郭露, 刘金明, 李浩, 古少鹏, 金亚美. 日本血吸虫ELAV-like 2基因的克隆、表达及功能分析[J]. 畜牧兽医学报, 2018, 49(9): 1987-1997.

ZHANG Yuan-yuan, XU Rong, HE Chuan-chuan, LI Xiao-chun, CHENG Gui-feng, GUO Lu, LIU Jin-ming, LI Hao, GU Shao-peng, JIN Ya-mei. Cloning,Expression and Functional Analysis of ELAV-like 2 Gene of Schistosoma japonicum[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1987-1997.

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日本血吸虫ELAV-like 2基因的克隆、表达及功能分析

Cloning,Expression and Functional Analysis of ELAV-like 2 Gene of Schistosoma japonicum

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