日本血吸虫胰凝乳蛋白酶样蛋白基因功能的初步研究  

Preliminary Genetic and Functional Analyses of Schistosoma japonicum Chymotrypsin-like Protease

作　　者：柴日奕 王吉鹏[1] 李健[1] 张瑞祥[1] 李青[1] 刘牧[1] 辛玥[1] 徐斌[2] 胡薇[1,2] 

机构地区：[1]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,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第4期251-257,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81271867)~~

摘　　要：目的探索日本血吸虫胰凝乳蛋白酶样蛋白(chymotrypsin-like protease)在终宿主体内的基因表达、定位和潜在功能,评估该蛋白在小鼠抗血吸虫感染中的免疫保护力。方法利用软件分析日本血吸虫胰凝乳蛋白酶样蛋白(Sj CTRL)基因(Gen Bank登录号为FN317561.1)的理化特性,及与其他物种同源基因的种系发生关系。采用整体原位杂交法定位Sj CTRL基因转录本在d26成虫中的表达部位。利用实时定量荧光PCR方法监测感染后14、18、22和26 d等4个发育时期雌雄虫的Sj CTRL基因转录水平。制备Sj CTRL-ds RNA,采用体外浸泡法对d26合抱雌雄虫进行RNA干扰,并利用共聚焦显微镜观察被干扰虫体的形态学变化。设计特异性引物扩增去除跨膜结构的编码基因序列,并克隆至p ET-28a原核表达载体,转化大肠埃希菌(E.coli)BL21(DE3)后进行原核表达。将纯化重组蛋白免疫C57BL/6小鼠,用尾蚴(40±2条/鼠)攻击感染进行动物保护性实验。同时设对照组。感染后35 d收集虫体和肝脏,统计减虫率、每克肝组织减卵率。结果原位杂交定位结果显示,该基因转录本位于雌虫后段肠道。Sj CTRL基因仅在d26雌虫体内有较高的转录本丰度。Sj CTRL-ds RNA干扰后雌虫相应基因转录本的相对表达量降至25.7%(P<0.05),但未有可观察到的形态学变化。构建了p ET-28a/Sj CTRL重组质粒,诱导表达获得不可溶重组蛋白。免疫保护试验结果显示,减虫率为25.4%,减卵率为80.5%。结论初步探明日本血吸虫胰凝乳蛋白酶样蛋白基因仅在d26雌虫的后段肠道高转录,体外干扰可降低Sj CTRL基因的转录本水平,该蛋白可一定程度上减少感染日本血吸虫的C57BL/6小鼠的成虫数和肝脏虫卵数。Objective To investigate the gene expression, localization and potential functions of Schistosoma japonicum chymotrypsin-like protease（SjCTRL） in the host, and evaluate its potential immune-protection efficacy against S. japonicum infection in mice. Methods The physicochemical properties of SjCTRL and its phylogenetic relationship with homologous genes from other species were analyzed with bioinformatic software. The distribution of SjCTRL transcripts in 26-day-old worms was investigated using whole-mount in situ hybridization. The transcriptional levels of SjCTRL in male and female worms at four development stages（14, 18, 22, and 26 days after infection） were measured with quantitative real-time PCR. The SjCTRL-dsRNA was prepared and used to induce RNA interference（RNAi） in 26-day-old worms via soaking in vitro, and confocal microscopy was used to observe the morphological changes of worms after RNAi. Primers were designed to amplify the encoding sequence（excluding the transmembrane region） from the S. japonicum cDNA. The truncated gene was subcloned into the pET-28a plasmid, transformed into E. coli BL21（DE3） for expression. Mice were immunized with the purified recombinant protein and challenged with cercariae. The worms and mouse liver were collected on day 35 after the challenge, and the worm-reduction rate and egg-reduction rate were calculated. Results The in situ hybridization results showed that SjCTRL mRNA was located in the posterior segment of intestinal tract of female worms, and having abundence only in 26-day-old female worms. After RNAi with SjCTRL-dsRNA, the mRNA expression was reduced to 25.7% （P〈0.05）, without significant morphological changes. Using the recombinant plasmid pET-28a/SjCTRL, expression of insoluble SjCTRL protein was induced. Mice immunized with this protein gained a worm-reduction rate of 25.4% and an egg-reduction rate of 80.5% in liver after being challenged with cercariae. Conclusion This study proves a high transcriptional level of SjCTRL in

关 键 词：日本血吸虫 胰凝乳蛋白酶样蛋白 原位杂交 RNA干扰 免疫保护 

分 类 号：R383.24[医药卫生—医学寄生虫学]