细粒棘球绦虫原头节抗原分子Eg-08002的克隆、表达及免疫反应性分析

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (6): 559-562.

细粒棘球绦虫原头节抗原分子Eg-08002的克隆、表达及免疫反应性分析

杨慧^1,², 赵殷奇^1,^2,³, 李子华^1,^2,³, 赵嘉庆^2,⁴, 王浩^2,⁴, 王娅娜⁵, 朱明星^1,^2,³, 赵巍^1,^2,^*()

1 宁夏医科大学,科技中心
2 宁夏医科大学,包虫病实验室
3 宁夏医科大学,基础医学院
4 宁夏医科大学,基础医学院病原与免疫教研室
5 宁夏医科大学,基础医学院遗传与细胞生物教研室,银川 750004

收稿日期:2017-07-27 出版日期:2017-12-30 发布日期:2018-01-10
通讯作者: 赵巍
基金资助:
国家自然科学基金（No.81360249）;宁夏自然科学基金（No.NZ17208）;宁夏医科大学校级课题（No.XM2016015）

Cloning, expression, and immunoreactivity analysis of antigen Eg-08002 from protoscoleces of Echinococcus granulosus

Hui YANG^1,², Yin-qi ZHAO^1,^2,³, Zi-hua LI^1,^2,³, Jia-qing ZHAO^2,⁴, Hao WANG^2,⁴, Ya-na WANG⁵, Ming-xing ZHU^1,^2,³, Wei ZHAO^1,^2,^*()

1Ningxia Medical University, 1 The Sci-technology Center
2 Echinococcosis Laboratory
3 Institute of Basic Medical College
4 Department of Pathogenic Biology and Medical Immunology, School of Basic Medicine
5 Medical Genetics and Cell Biology Department, School of Basic Medicine, Yinchuan 750004, China

Received:2017-07-27 Online:2017-12-30 Published:2018-01-10
Contact: Wei ZHAO
Supported by:
Supported by the National Natural Science Foundation of China（No.81360249）, the Natural Science Foundation of Ningxia（No.NZ17208） and the Foundation of Ningxia Medical University（No.XM2016015）

摘要/Abstract

摘要：

目的筛选细粒棘球蚴（Echinococcus granulosus, Eg）原头节抗原分子,并进行克隆、表达及免疫反应性分析。方法在对细粒棘球绦虫六钩蚴和原头节阶段表达的转录组测序数据进行差异分析的基础上,筛选出原头节阶段特异性表达的抗原分子。PCR扩增后克隆至pET28a载体,构建原核表达载体pET28a-Eg-08002,转化至大肠埃希菌（Escherichia coli）JM109,异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,抗His标签镍亲和层析柱纯化重组蛋白rEg-08002,快速免染聚丙烯酰胺电泳分析表达产物;蛋白质印迹（Western blotting）分析rEg-08002的免疫反应性。ELISA分析重组蛋白rEg-08002与细粒棘球蚴病患者血清和健康人血清的免疫反应性。结果筛选出了原头节中高表达的抗原分子Eg-08002,大小为612 bp,构建原核表达载体pET28a-Eg-08002。快速免染聚丙烯酰胺电泳和Western blotting分析结果显示重组蛋白rEg-08002在E.coli JM109中获得高效表达,在相对分子质量（M_r）约为23 000处可见重组蛋白rEg-08002条带,主要以包涵体形式存在,rEg-08002可被感染棘球蚴的小鼠血清识别。ELISA分析结果表明,12份细粒棘球蚴病患者血清和12份健康人血清的平均吸光度（A₄₅₀）值分别为1.245 ± 0.265和0.469 ± 0.006,差异具有统计学意义（P < 0.05）。结论筛选出了原头节中高表达的抗原分子Eg-08002,获得的重组蛋白rEg-08002具有较好的免疫反应性。

关键词: 细粒棘球蚴, 克隆, 表达, 免疫反应性

Abstract:

Objective To clone, express and analyze the immuno-reactivity of Eg-08002, an antigen screened out from protoscolex-stage Echinococcus granulosus.Methods Based on a previous study of transcriptome sequencing at the protoscolex phase and oncosphere, the protoscolex stage specific antigen Eg-08002 was identified and amplified by PCR and cloned into vector pET28a to construct recombinant plasmid pET28a-Eg-08002, which was then transformed into Escherichia coli JM109 for expression under the induction of IPTG.The recombinant rEg-08002 protein was purified with His-tag Ni-superflow affinity chromatography, and verified by rapid non-staining polyacrylamide electrophoresis.Immunoreactivity of rEg-08002 was analyzed by Western blotting, which was then assessed with sera from 12 patients with cysitic echinococcosis and 12 healthy individuals by ELISA.Results High expression of Eg-08002 was detected in protoscolex, with a length of 612 bp.The recombinant pET28a-Eg-08002 was constructed.Results of rapid polyacrylamide gel electrophoresis and Western blotting indicated that the recombinant protein rEg-08002 was highly expressed in E.coli JM109 in a predominant form of inclusion bodies, with an M_r of 23000, and was recognized by sera from mice with cystic echinococcosis.ELISA results showed that the average A₄₅₀ values with sera from the patients and healthy individuals were 1.245 ± 0.265 and 0.469 ± 0.006, respectively, with statistical difference（P < 0.05）.Conclusion The antigen Eg-08002 was identified with high expression in the protoscolex phase with a promising immunoreactivity as revealed by Western blotting and ELISA

Key words: Echinococcus granulosus, Cloning, Expression, Immunoreactivity

中图分类号:

R383.3

杨慧, 赵殷奇, 李子华, 赵嘉庆, 王浩, 王娅娜, 朱明星, 赵巍. 细粒棘球绦虫原头节抗原分子Eg-08002的克隆、表达及免疫反应性分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 559-562.

Hui YANG, Yin-qi ZHAO, Zi-hua LI, Jia-qing ZHAO, Hao WANG, Ya-na WANG, Ming-xing ZHU, Wei ZHAO. Cloning, expression, and immunoreactivity analysis of antigen Eg-08002 from protoscoleces of Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 559-562.

图/表 3

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