日本血吸虫SjELAV-like 1的克隆、表达及功能分析

Cloning, Expression and Function Analysis of Schistosoma Japonicum ELAV-like 1

【目的】克隆和表达日本血吸虫胚胎致死异常视觉基因1(Sj ELAV-like 1),分析其在日本血吸虫体内的表达情况及定位,探究该基因对日本血吸虫形态和生殖发育的影响。【方法】利用RACE技术扩增5′/3′末端,获得Sj ELAV-like 1基因序列提交至NCBI,Gene Bank登录号:MG515727,构建该序列的重组表达质粒p ET28-Sj ELAV-like 1,并在大肠杆菌BL21中利用IPTG诱导表达,收集不同诱导时间的菌体,SDS-PAGE法进行蛋白电泳。利用His镍柱亲和层析法纯化大量表达所得重组蛋白,用纯化重组蛋白免疫小鼠制备多克隆抗体,用于Western blotting分析虫体内该天然蛋白的免疫原性,以及间接免疫荧光检测该蛋白在虫体内的分布情况。小鼠腹部贴片法感染日本血虫尾蚴,收集不同发育阶段混合虫体和合抱分开的雌、雄虫体,以单钉螺逸出的尾蚴感染小鼠获得25 d单性雌、雄虫体,通过荧光定量PCR分析该基因的表达情况。通过尾静脉注射小干扰RNA(si RNA)于感染小鼠体内,使该基因表达沉默,筛选最佳干扰效果的si RNA,用于长期RNA干扰(RNAi)试验,分析其对日本血吸虫雌虫产卵及卵胚孵化的影响。利用扫描电镜和透射电镜观察RNAi对虫体体被结构及雌、雄虫生殖器官的影响。【结果】获得的1 797 bp的Sj ELAV-like 1基因序列含有poly A尾,其中编码序列为1 533 bp,编码510个氨基酸。重组质粒p ET28-Sj ELAV-like 1在诱导4 h时表达量最高,主要以包涵体的形式存在,利用纯化蛋白获得了优质多抗,获得的重组蛋白具有良好的免疫原性,经分析发现Sj ELAV-like 1蛋白主要分布在虫体体被。荧光定量分析发现Sj ELAV-like 1在不同时期雌、雄虫体内均有表达,在雄虫体内表达稳定,但在雌虫体内随着发育成熟其表达水平呈下降趋势;在不同感染状态的雌、雄虫体内,与正常发育雌虫相比,该基因在发育阻遏雌虫体内高表达,而在雄虫体内无明显差异。RNAi试验中,与NC组相比,长期干扰组小鼠肝减卵率、雌虫对肝卵贡献减少率和卵胚减孵化率分别为58.27%(P<0.05)、40.59%(P<0.01)和74.58%(P<0.01);电镜观察发现,干扰组虫体体被结构改变,雄虫体表粘附的泡状物消失,立体的褶脊结构塌陷,整齐排列的条索状皮脊变的疏松,网状结构消失,雌虫体壁表面组织间隙增宽,分布于其上的体棘减少、变钝,雄虫精母细胞数目减少,其内的染色质减少,细胞肿大,细胞间隙增宽,雌虫卵黄细胞中卵黄球减少,其中包含的卵黄滴也减少,内质网肿大,皮质颗粒排列散乱。【结论】成功克隆和表达了在日本血吸虫发育阻遏雌虫中高表达的部分Sj ELAV-like 1基因序列,且该基因主要在虫体体被表达。该基因沉默使肝脏荷卵数、雌虫对肝卵贡献率和卵胚孵化率均显著降低,并改变虫体体被结构,抑制生殖腺的正常发育,提示该基因对日本血吸虫的生殖发育有重要的作用。

【Objective】 Cloning and expressing of Schistosoma japonicum embryonic lethal abnormal vision like 1（Sj ELAVlike 1） gene and analyzing the expression status in different stages and at different infected conditions as well as its distributions in the worms of Schistosoma japonicum（S. japonicum）. This study also intend to explore the effects of Sj ELAV-like 1 on the morphology and reproductive development of S. japonicum.【Method】 The RACE technique was used to amplify the 5′/3′ end of the Sj ELAV-like 1 gene, the obtained sequence was submitted to NCBI, Gene Bank accession number： MG515727. The recombinant plasmid p ET28-Sj ELAV-like 1 was constructed and expressed in E.coli BL21 by IPTG induction, then the products were collected at different time. The SDS-PAGE was used for protein analysis. His-tag nickel column affinity chromatography was used to purify the recombinant protein. The purified recombinant protein was used to immune mouse to obtain the polyclonal antibody, which was used to analyze the immunogenicity of the recombinant protein by western blotting and detect the distributions of Sj ELAV-like 1 protein in S. japonicum by indirect immunofluorescence assay. The mice infected by cercaria of S. japonicum in the abdomen were used to collect mixture and the females and males separated by paired worms at different stages. The mice infected by cercaria escaped from the single nails were used to collect the unisexual worms at the 25 th day. The expression level of Sj ELAV-like 1 were detected by quantitative real-time PCR（q RT-PCR）. The gene silence was performed by tail vein injection of small interference RNA（si RNA） in infected mice. The long-term RNA interference（RNAi） assay was performed to analyze the effects of Sj ELAV-like 1 gene silence onthe spawning and egg hatching of females. The tegument structures and reproductive organs of the females and males of S. japonicum were observed by scanning electron microscopy and transmission electron microscopy after RNAi.【Result】In this study, a 1 797 bp sequence of Sj ELAV-like 1 was obtained, whose coding region was 1 533 bp encoding 510 amino acids. The expression of the recombinant plasmid reached its highest after 4 h of IPTG induction and it mainly existed in the form of inclusion bodies. High quality polyclonal antibody was obtained against the purified protein. Sj ELAV-like 1 protein was mainly located in the tegument of the worms. Sj ELAV-like 1 was expressed in all stages of S. japonicum and the expression was stable in males but gradually became less in females as the maturing of the worms. Expression of Sj ELAV-like 1 was higher in development impaired females compared with the normal worms. In the RNAi assay, the long-term interference group showed that liver egg burden, liver egg burden by per female and egg hatching rate in interference group were reduced by 58.27%（P〈0.05）, 40.59%（P〈0.01） and 74.58%（P〈0.01） compared with NC group, respectively. Electron microscopy observation found that the tegument structures of the worms in interference group were obviously different from that in NC group. The surface bubble adhere to the surface of males disappeared, the three-dimensional fold ridge collapsed, the funicular surface ridge arranged loosely and the network structure of the body wall disappeared. The body surface gap increased and the spines on the body wall of the female were dull. The number of spermatocyte in the males was decreased and the intracellular chromatin was reduced, while the cells were swollen and the intercellular space was widen. In females of S. japonicum the vitelline globules in vitelline cells were decreased, in which the vitelline droplets were also reduced, the endoplasmic reticulum was swollen and the cortical particles were scattered.【Conclusion】Part sequence of Sj ELAV-like 1 was successfully cloned and expressed, which was robustly expressed in development impaired females. Sj ELAV-like 1 was mainly expressed in the tegument of S. japonicum. Sj ELAV-like 1 silence led to decrease of liver egg burden, liver egg burden by per female and egg hatching rate. Sj ELAV-like 1 silence also altered the tegument structures of the worms and hindered its development of reproductive glands. These all suggested that Sj ELAV-like 1 play an important role in the development of the reproduction of S. japonicum.