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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。
OBJECTIVE: To clone and prokaryotic express the newly discovered CsMLP from Clonorchis sinensis, and to understand the immunological function of its recombinant product. Methods The bioinformatics analysis software was used to analyze the sequence characteristics and basic physical and chemical characteristics of CsMLP. The CsMLP gene was cloned into the prokaryotic expression plasmid pET-28a (+), induced and expressed, and purified by nickel ion metal chelating affinity chromatography column. The antiserum was obtained by immunizing SD rats with purified CsMLP protein. Immunoreactivity and immunogenicity were analyzed by Western blotting. The localization of CsMLP in adult Clonorchis sinensis was observed by immunofluorescence staining. Results The cDNA sequence was 900 bp in length and encoded 190 amino acids with Calponin domain. The results of PCR, double enzyme digestion and DNA sequencing indicated that the recombinant plasmid pET-28a (+) - CsMLP was constructed successfully. SDS-PAGE showed that the target gene Highly expressed in Escherichia coli BL21 / DE3, the molecular weight of 21300Mr; obtained by affinity chromatography of high purity protein; recombinant protein can be immune SD rat serum, Clonorchis sinensis infected SD rat serum identification ; CsMLP is mainly located in the mouth of the worm body rich in muscle tissue, abdominal suction disk, pharynx, in the epidermis, intestinal wall distribution. Conclusion CsMLP can express efficiently in prokaryotic expression system and has immunogenicity. The majority of CsMLP is found in muscle-rich tissues of Clonorchis sinensis.