简单异尖线虫肌钙蛋白类过敏原基因的克隆表达及免疫特性分析

Cloning,expression and immunologic identification of troponin-like allergen gene of Anisakis simplex

为研究简单异尖线虫肌钙蛋白类过敏原的免疫特性,本研究提取了线虫总RNA,逆转录合成cDNA,PCR扩增肌钙蛋白类过敏原(Anis1)基因,克隆入pMD18-T载体,测序正确后将anis1基因亚克隆入带有组氨酸标签的表达载体pET-30a,转入大肠杆菌BL21中,经异丙基硫代-β-D-半乳糖苷诱导,表达目的蛋白。通过Ni2+亲和层析柱对重组蛋白进行纯化,融合蛋白采用Western blotting分析。将纯化的Anis1重组蛋白免疫小鼠,分离血清,ELISA检测不同时期血清抗体效价。酶切和DNA测序结果显示重组质粒构建成功,并诱导出重组Anis1蛋白,重组蛋白经Western blotting分析证实为目的蛋白,纯化蛋白Anis1免疫小鼠后,与对照组相比,能产生较高的抗体水平。Western blotting分析显示,重组蛋白Anis1能被免疫血清识别,说明简单异尖线虫重组蛋白Anis1具有较好的免疫原性和免疫反应性。

In order to examine the immunological characteristics of troponin-like allergen （ Anisl ） of Aniakis simplex, total RNA was ex- tracted from A. simplex and reversely transcribed into cDNA. The anisl gene was amplified by PCR and cloned into pMD18-T vector for se- quencing, and then subcloned into the expression vector pET-30a with （His） 6-tag. The recombinant plasmid was transformed into Esche- richia coli BL21 and expressed with the induction of IPTG. The recombinant protein was identified by Western blotting. The mice were im- munized with the purified Anisl antigen. Serum samples were collected and the antibody titer was analyzed by ELISA. The results of enzyme digestion and DNA sequencing showed that the recombinant plasmids were successfully constructed. Western blotting analysis demonstrated the expression of recombinant Anisl protein. Compared with adjuvant group, mice immunized with purified protein Anisl produced a higher level of IgG. The anti-Anisl serum could recognize the recombinant Anisl protein. It suggested that the recombinant protein Anisl possessed strong immunogenicity and immunoreactivity.