华支睾吸虫钙调理蛋白的克隆表达、组织定位及功能的初步研究  

Cloning, immunolocalization and basic function analysis of the Calponin gene from Clonorchis sinensis

作　　者：李然[1,2] 余新炳[1,2] 陈庭金[1,2] 邓传欢[1,2] 王乐旬[1,2] 李文芳[1,2] 黄艳[1,2] 徐劲[1,2] 

机构地区：[1]中山大学中山医学院寄生虫学教研室,广东广州510080 [2]中山大学热带病防治研究教育部重点实验室

出　　处：《中国病原生物学杂志》2013年第5期411-416,共6页Journal of Pathogen Biology

基　　金：国家973发展计划项目(No.2010CB530000);国家自然科学基金项目(No.81101270;81171602)

摘　　要：目的克隆和原核表达华支睾吸虫钙调理蛋白(CsCALP),并初步研究其功能及免疫学意义。方法利用生物信息学相关软件分析CsCALP蛋白的基本理化特征和三维结构;将CsCALP基因克隆到原核表达质粒pET-28a(+)中,诱导表达CsCALP蛋白并用镍离子亲和层析柱进行纯化;通过凝胶覆盖(Gel overlay)试验鉴定纯化的CsCALP蛋白与F-Actin的结合能力;使用纯化的CsCALP蛋白免疫SD大鼠,获得抗血清,通过Western blot分析其免疫学特性;应用免疫荧光化学法观察华支睾吸虫CsCALP蛋白在成虫的定位。结果 CsCALP基因编码序列长度为1086bp,编码361个氨基酸,理论分子质量单位为39.7708ku,具有1个calponin homology结构域和5个calponin-like repeat结构域;该基因在大肠埃希菌BL21/DE3中可被表达,纯化的重组蛋白能被感染华支睾吸虫的大鼠血清识别;CsCALP蛋白主要定位于虫体富含肌肉组织,包括口、腹吸盘、咽部以及皮下肌层;Gel-overlay试验显示CsCALP蛋白能与F-Ac-tin结合。结论构建的pET-28a(+)-CsCALP能在大肠埃希菌中表达可溶性CsCALP蛋白。该蛋白具有良好的抗原活性,主要分布在华支睾吸虫成虫肌肉组织中,可能为分泌排泄抗原的组分之一。Objective To clone and express the gene encoding Clonorchis sinensis calponin and study its basic function and immunolocalization. Methods The structure and physical and chemical parameters of C. sinensis calponin were analyzed using bioinformatics software. The open reading frame of Calponin was amplified by PCR and cloned into a prokaryotic expression vector, pET-28a （＋）, and then expressed in E. coli BL21. The recombinant protein was purified by Ni-DNA affinity chromatography and identified by immunoblotting. A gel-overlay experiment was used to demonstrate the combination of Calponin and F-Actin. The recombinant protein was used to raise antibodies for immunolocalization. Results The complete cDNA sequence of Calponin is 1,086 bp in length and encodes 361 amino acids. The theoretical molecular weight of Calponin is 39,770.8 Da. One calponin homology domain and five calponin-like repeats were found in Calponin. The Calponin gene was expressed in E.coli BL21. The recombinant protein reacted with rat sera infected with C. sinensis. The results of immunolocalization indicated that C. sinensis Calponin was located at the oral and ventral suckers and pharynx of adult flukes. Gel-overlay results revealed that Calponin binds to F-Actin. Conclusion C. sinensis Calponin is highly expressed in E. coli BL21 as a soluble protein. The recombinant protein had satisfactory antigenicity. Calponin is primarily located in the fluke＇s muscle layer and may be one of the components of ESA of C. sinensis.

关 键 词：华支睾吸虫 钙调理蛋白 克隆 免疫组化 

分 类 号：R383.22[医药卫生—医学寄生虫学]