一种华支睾吸虫亲肌素样基因的克隆、表达与免疫学功能初步研究

目的对新发现的华支睾吸虫亲肌素样蛋白(CsMLP)进行克隆、原核表达,初步了解其重组产物的免疫学功能。方法应用生物信息学分析软件,分析CsMLP的序列特点和基本理化特征;将CsMLP基因克隆到原核表达质粒pET-28a(+)中,诱导表达并用镍离子金属螯合剂亲和层析柱进行纯化,用纯化的CsMLP蛋白免疫SD大鼠获得抗血清;用Westernblot进行免疫反应性及免疫原性分析;应用免疫荧光方法观察CsMLP在华支睾吸虫成虫的定位。结果该cDNA序列全长为900bp,编码190个氨基酸,具有Calponin功能域;PCR、双酶切及DNA测序结果均表明pET-28a(+)-CsMLP重组质粒构建成功;SDS-PAGE结果表明目的基因在大肠杆菌BL21/DE3中获得高效表达,分子量为21300Mr;经亲和层析获得了高纯度蛋白;重组蛋白可被其免疫的SD大鼠血清、感染了华支睾吸虫的SD大鼠血清识别;CsMLP主要定位于虫体富含肌肉组织的口、腹吸盘、咽部,在表膜、肠壁也有分布。结论 CsMLP可在原核表达系统中呈现高效的可溶性表达,具有免疫原性,其主要分布在华支睾吸虫肌肉丰富的组织。

Identification,characterization and immunolocalization of myophilin-like:a muscle-specific antigen of Clonorchis sinensis

Objective To clone and expressed the gene encoding Clonorchis sinensis myophilin-like protein(CsMLP) and study the antigenicity of the recombinant protein.Methods The structural and functional characteristics of CsMLP were analyzed by using bioinformatic software.The open reading frame of CsMLP was amplified by PCR and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21.The recombinant protein was purified by NI-DNA affinity chromatography and detected by SDS-PAGE.The immune reactivity and antigenicity of CsMLP were analyzed by Western blot.Results It was demonstrated that the complete cDNA sequence of CsMPL is 900 bp in length encoding 190 amino acids.The theoretical molecular weight of CsMPL is 21 300 Mr.The results of SDS-PAGE and western blotting show that the molecular weight of expressed fusion protein was around 20 000 Mr.CsMLP can be detected with the sera from Clonorchis sinensis infected rat and the sera from rat immunized with recombinant protein.CsMLP is mostly distribute at the oral sucker,acetabulum,pharynx of Clonorchis sinensis.Conclusions Recombinant CsMLP is highly expressed in E.coli BL21 as a soluble protein.The recombinant protein is antigenic.CsMLP expresses in the muscular tissues of the adult worm.