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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

26卷

期数:

2020年03期

页码:

256-260

栏目:

技术与方法

出版日期:

2020-06-10

文章信息/Info

Title:

Recombinant expression, identification and immunoglobulin E binding analysis of tropomyosin Pen a 1

文章编号:

1006-8147(2020)03-0256-05

作者:

刘甫¹; 李军普¹; 李绍深²; 李智伟¹; 孔德玉³; 白虹⁴; 李会强¹

（1.天津医科大学医学技术学院，天津300203；2.天津市中医药研究院附属长征医院检验科，天津300120；3.天津市港口医院检验科，天津300456；4.天津医科大学基础医学院免疫学系，天津300070）

Author(s):

LIU Fu¹;  LI Jun-pu¹;  LI Shao-shen²;  LI Zhi-wei¹;  KONG De-yu³;  BAI Hong⁴;  LI Hui-qiang¹

（1. Medical Technology College， Tianjin Medical University，Tianjin 300203，China； 2. Department of Clinical Laboratory，Tianjin Institute of Traditional Chinese Medicine, Tianjin 300120，China；3.Department of Clinical Laboratory，Tianjin Port Hospital，Tianjin 300456，China； 4.Department of Immunology, School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070， China)

关键词:

贝类过敏; 重组过敏原; 原肌球蛋白（Pen a 1）; 免疫球蛋白E

Keywords:

shellfish allergy; recombinant allergen; tropomyosin (Pen a 1); IgE

分类号:

R446.61

DOI:

-

文献标志码:

A

摘要:

目的：在大肠杆菌中重组表达贝类主要过敏原—原肌球蛋白(Pen a 1)，并且评估其IgE结合活性。方法：首先基于Pen a 1 的已知序列人工合成Pen a 1 基因，其次将其与质粒pET-28a连接以构建重组表达质粒pET-28a(+)-Pen a 1，经测序、酶切鉴定后导入表达宿主E. coli BL21（DE3）细胞中。通过IPTG诱导，重组表达Pen a 1蛋白，镍柱纯化表达产物，并采用SDS-PAGE电泳及质谱技术鉴定所得重组蛋白。最后应用贝类过敏患者采用LICA技术检测该重组蛋白的IgE结合活性。结果：PCR结果表明，目的基因全长为930 bp，与理论预测值一致。SDS-PAGE电泳结果显示，目的蛋白在宿主菌内以可溶性蛋白的形式高效表达，分子量约为 36 kD ，大小与理论值相符。质谱鉴定结果进一步说明所得的重组蛋白为原肌球蛋白Pen a 1。免疫分析结果表明该蛋白具有较高的IgE结合能力。结论：成功表达、纯化出有良好免疫学活性的重组原肌球蛋白Pen a 1，为进一步研究Pen a 1在贝类动物如虾类过敏的诊断和治疗中作用奠定基础。

Abstract:

Objective: To clone and express Pen a 1 as a major allergen from Penaeus aztecus in E.coli, and to analyze its immunoglobulin E (IgE)binding ability. Methods: The gene coding Pen a 1 protein sequences were synthesized and cloned into the Bam HI and Hind III sites of pET28a (+) expression vector. After sequencing and identified by restriction endonuclease digestion, the verified pET-28a(+)-Pen a 1 was transformed into E. coli BL21 cells. The recombinant expression of Pen a 1 was induced by IPTG. The recombinant protein was purified using Ni+-NTA affinity column chromatography, characterized by sodium dodecyl sulphate SDS-PAGE and MS, and tested by LICA for IgE reactivity with sera from individuals with shellfish allergy. Results: PCR analysis showed that the size of the target gene fragment was about 930 bp, which was consistent with the theoretical predictions. SDS-PAGE showed that the recombinant protein was highly expressed in the form of soluble protein in E. coli, and its molecular weight was about 36 kD, which was in accordance with the theoretical value. The results of mass spectrometry further revealed that the expression product was tropomyosin Pen a 1. Immunological analysis indicated the protein had high IgE binding ability. Conclusion: Recombinant tropomyosin Pen a 1 with good immunogenicity was successfully produced, which laid the foundation for further study on the role of Pen a 1 in the diagnosis and treatment of allergies including shrimp allergy.

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相似文献/References:

备注/Memo

备注/Memo:

基金项目 国家自然科学基金资助项目（81772259）；天津医科大学校内科研基金资助项目（2110/2JY018）
作者简介 刘甫（1985-），男，助理实验师，硕士在读，研究方向：免疫学；通信作者：白虹，E-mail：Hongbai25@163.com；李会强，E-mail：lihuiqiang1965@163.com。

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更新日期/Last Update: 2020-06-13