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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 prokaryotic express the calcium-regulated protein (CsCALP) of Clonorchis sinensis and to study its function and immunological significance. Methods The basic physicochemical properties and three-dimensional structure of CsCALP protein were analyzed by bioinformatics software. The CsCALP gene was cloned into prokaryotic expression plasmid pET-28a (+) and CsCALP protein was induced and purified by nickel ion affinity chromatography. Gel overlay assay was used to identify the binding capacity of purified CsCALP protein to F-Actin. The purified SDCs were immunized with purified CsCALP protein to obtain antiserum. The immunological characteristics were analyzed by Western blot. Immunofluorescence Clonorchis sinensis CsCALP protein localization in adults. Results The coding sequence of CsCALP gene was 1086 bp in length and encoded 361 amino acids. The theoretical molecular mass unit was 39.7708 ku, with one calponin homology domain and five calponin-like repeat domains. The gene was expressed in Escherichia coli BL21 / DE3 CsAALP protein was mainly localized in the muscle-rich tissues of the parasites, including the mouth, abdominal sucker, pharynx, and subcutaneous muscular layer. The Gel-overlay test CsCALP protein was shown to bind to F-Ac-tin. Conclusion The constructed pET-28a (+) - CsCALP can express soluble CsCALP protein in Escherichia coli. The protein has good antigen activity, mainly distributed in muscle tissue of adult Clonorchis sinensis and may be one of the components secreting antigen.