细粒棘球绦虫原头节抗原分子Eg-01883的克隆、表达及免疫原性分析

中国寄生虫学与寄生虫病杂志

细粒棘球绦虫原头节抗原分子Eg-01883的克隆、表达及免疫原性分析

赵殷奇1,2,3，李子华1,2,3，王浩2,4，朱明星1,2,3，牛楠2,3,5，王娅娜6，赵嘉庆2,4，李娜2,3,5，佟雪琪2,3,5，宋佳卉1,2,3，赵巍2,3*

宁夏医科大学，1基础医学院；2包虫病实验室；3科技中心；4基础医学院病原与免疫教研室；5临床学院检验系；6基础医学院遗传与细胞生物教研室，银川750004

出版日期:2016-06-30 发布日期:2016-10-28

Cloning, Expression and Immungenicity Analysis of Antigen Eg-01883 Screened from Protoscoleces of Echinococcus granulosus

ZHAO Yin-qi1,2,3，LI Zi-hua1,2,3，WANG Hao2,4，ZHU Ming-xing1,2,3，NIU Nan2,3,5， WANG Ya-na6，ZHAO Jia-qing2,4，LI Na2,3,5，TONG Xue-qi2,3,5，SONG Jia-hui1,2,3，ZHAO Wei2,3*

Ningxia Medical University，1 Institute of Basic Medical College；2 Echinococcosis Laboratory；3 The Sci-technology Center；4 Department of Pathogenic Biology and Medical Immunology，School of Basic Medicine；5 Department of Laboratory；6 Medical Genetics and Cell Biology Department，School of Basic Medicine，Yinchuan 750004，China

Online:2016-06-30 Published:2016-10-28

摘要/Abstract

摘要： 目的筛选细粒棘球蚴原头节抗原分子Eg-01883，并进行克隆、表达及免疫原性分析，为寻找棘球蚴病特异性诊断抗原提供依据。方法分析已发表的细粒棘球绦虫mRNA测序数据，筛选六钩蚴中不表达、原头节中高表达的抗原分子Eg-01883。提取细粒棘球蚴原头节总RNA，用RT-PCR对目的基因Eg-01883进行克隆，重组构建原核表达载体pET28a-Eg-01883，异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达并纯化目的重组蛋白rEg-01883。ICR小鼠随机分为3组（每组12只），免疫组小鼠的背部皮下多点注射rEg-01883，2周后加强免疫1次（剂量均为10 μg，100 μl/只）；佐剂组注射福氏佐剂和PBS，空白对照组不作任何处理。分别于免疫前，首次免疫后1、2、4周每组小鼠尾静脉采血，免疫后6周进行去眼球采血。用ELISA检测3组小鼠免疫后不同时间点的血清特异性IgG抗体水平，及细胞因子白介素4（IL-4）和γ干扰素（IFN-γ）水平。蛋白质印迹（Western blotting）分析重组蛋白rEg-01883的免疫原性。结果筛选出在细粒棘球蚴原头节中高表达的抗原分子Eg-01883，克隆、表达和纯化后获得重组蛋白rEg-01883，该蛋白主要以包涵体形式存在。ELISA检测结果显示，用纯化后的重组蛋白rEg-01883免疫小鼠，可诱导产生特异性IgG抗体，免疫组小鼠的血清IgG抗体水平自首次免疫后1周开始上升，6周时达到最高水平（2.344±0.153），显著高于佐剂组（0.206 1±0.006）和空白对照组（0.241±0.01）（P<0.01）。首次免疫后6周，血清中的细胞因子IFN-γ和IL-4水平，免疫组分别为43.23 pg/ml和24.88 pg/ml，均显著高于佐剂组（21.77 pg/ml，13.27 pg/ml）和空白对照组（17.40 pg/ml，12.25 pg/ml）（P<0.05）。Western blotting分析结果显示，该重组蛋白rEg-01883抗原可被His-Tag标签抗体、免疫组小鼠血清、原头节继发感染的小鼠血清识别。结论筛选获得细粒棘球绦虫原头节中高表达的抗原分子Eg-01883，克隆、表达、纯化后的重组蛋白rEg-01883免疫ICR小鼠具有较好的免疫原性。

关键词: 细粒棘球绦虫, 原头节, 重组蛋白rEg-01883, 原核表达, 免疫原性

Abstract: Objective To screen for the Echinococcus granulosus 01883（Eg-01883） specifically expressed at the protoscolex period, clone and express this molecule as well as analyse its immunogenicity. Methods Eg-01883, which is highly expressed at the protoscolex period but not in oncosphere， was screened by analysing the published mRNA sequences of E. granuolosus. Total RNA of E. granuolosus was extracted, Eg-01883 was cloned by RT-PCR, and the recombinant plasmid pET28a-Eg-01883 was constructed. Expression of the recombinant protein rEg-01883 was induced by isopropyl-β-D-thiogalactoside （IPTG）. ICR mice were randomized into 3 groups （n=12 in each group）. Mice in the immunization group received subcutaneous injections of 10 μg rEg-01883 in 100 μl PBS emulsified in Freund’s adjuvant at multiple sites, followed by immune enhancement after 2 weeks. Mice in the adjuvant group were injected with PBS and adjuvant. Mice in the control group received no treatment. Blood was obtained through caudal vein before immunization, and at 1, 2, and 4 weeks after the first immunization, and through the eyeball at 6 weeks after immunization. Serum levels of IgG, IFN-γ and IL-4 were determined by ELISA. The immunogenicity of rEg-01883 was identified by Western blotting. Results Eg-01883 was screened, cloned, expressed and purified to obtain the recombinant protein rEg-01883, which mainly existed as the inclusion body. ELISA results showed that immunization with rEg-01883 induced production of specific IgG antibody. The serum IgG level in the immunization group increased from 1 week after the first immunization, peaked at 6 weeks（2.344±0.153）, which was significantly higher than those of the adjuvant group（0.206 1±0.006） and the control group （0.241±0.01）（P<0.01）. At 6 weeks after the first immunization, the serum levels of IFN-γ （43.23 pg/ml） and IL-4（24.88 pg/ml） in the immunization group were significantly higher than those in the adjuvant group（21.77 pg/ml, 13.27 pg/ml） and the control group（17.40 pg/ml, 12.25 pg/ml）（P<0.05）. Western blot showed that the recombinant protein rEg-01883 could be recognized by His-Tag antibodies, serum of immunized mice, and serum of mice with secondary infection. Conclusion The recombinant protein rEg-01883 shows good immunogenicity in ICR mice.

Key words: Echinococcus granulosus, Antigen rEg-01883, Prokaryotic expression, Immunogenicity

赵殷奇1,2,3，李子华1,2,3，王浩2,4，朱明星1,2,3，牛楠2,3,5，王娅娜6，赵嘉庆2,4，李娜2,3,5，佟雪琪2,3,5，宋佳卉1,2,3，赵. 细粒棘球绦虫原头节抗原分子Eg-01883的克隆、表达及免疫原性分析[J]. 中国寄生虫学与寄生虫病杂志.

ZHAO Yin-qi1,2,3，LI Zi-hua1,2,3，WANG Hao2,4，ZHU Ming-xing1,2,3，NIU Nan2,3,5，. Cloning, Expression and Immungenicity Analysis of Antigen Eg-01883 Screened from Protoscoleces of Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases.

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