中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3679-3682.doi: 10.3969/j.issn.1673-8225.2011.20.017

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

蛔虫过敏原ABA-1融合基因在大肠杆菌中的表达及鉴定

于超生1，文  忠1，邹泽红2，陈惠芳2，陶爱林2   

  1. 1南方医科大学珠江医院耳鼻咽喉-头颈外科，广东省广州市，510282
    2广州医学院第二附属医院过敏反应与临床免疫重点实验室，呼吸疾病国家重点实验室，广东省广州市510260
  • 收稿日期:2011-02-26 修回日期:2011-04-09 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 陶爱林，博士，教授，广州医学院第二附属医院过敏反应与临床免疫重点实验室，呼吸疾病国家重点实验室广东省广州市，510260 Aerobiologiatao@163.com
  • 作者简介:于超生★，男，1982年生，山东省泰安市人，汉族，南方医科大学在读硕士，主要从事耳鼻咽喉-头颈外科的临床研究工作。 ycs_9982@163.com 并列第一作者：文忠，男，1960年生，博士，2000年中南大学毕业，湖南省常德市人，汉组，教授，主要从事头颈肿瘤基础与临床及鼻科学实验与临床研究工作。 wenzhong60@ 163.com
  • 基金资助:

    课题受国家科技重大专项重大课题(2008ZX08011-005)及重点课题(2009ZX08011-004B)、国家自然科学基金项(30771240)和广州市教育系统科研创新学术团队(B94118)资助。

Expression and identification of chimeric protein truncated from Ascaris allergen ABA-1

Yu Chao-sheng1, Wen Zhong1, Zou Ze-hong2, Chen Hui-fang2, Tao Ai-lin2   

  1. 1Department of Otolaryngology-Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou  510282, Guangdong Province, China
    2Academic Key Laboratory of Allergy and Clinical Immunity, State Key Laboratory of Respiratory Diseases, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou  510260, Guangdong Province, China
  • Received:2011-02-26 Revised:2011-04-09 Online:2011-05-14 Published:2011-05-14
  • Contact: Tao Ai-lin, Doctor, Professor, Master’s supervisor, Academic Key Laboratory of Allergy and Clinical Immunity, State Key Laboratory of Respiratory Diseases, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong Province, China AerobiologiaTao@ 163. com
  • About author:Yu Chao-sheng★, Studying for master’s degree, Department of Otolaryngology- Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China ycs_9982@163.com Wen Zhong, Doctor, Professor, Department of Otolaryngology-Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China wenzhong60@163. com Yu Chao-sheng and Wen Zhong contributed equally to this paper.
  • Supported by:

    Great Project from the Major Program of National Science and Technology of China, No. 2008ZX08011-005*; Key Project from the Major Program of National Science and Technology of China, No. 2009ZX08011-004B*; the National Natural Science Foundation of China, No. 30771240*; Program for Educational & Scientific Innovation Team of Guangzhou City, No. B94118* 

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摘要：

背景：利用蛔虫基因融合技术，可将过敏原ABA-1的主要基因进行融合。
目的：利用大肠杆菌表达系统重组表达蛔虫主要过敏原ABA-1融合蛋白。
方法：从Gene Bank和Protein Database中获取蛔虫主要过敏原ABA-1基因和蛋白序列，选定其中的ABA-1B1，ABA-1A2与ABA-1A1基因重组为融合基因BAA，经密码子优化后，全基因合成目的基因BAA并构建于表达载体PET-44a上，经KCM法转化入大肠杆菌JM109中进行克隆，PET-44a/BAA经NdeⅠ和PstⅠ双酶切及DNA测序正确后，转化入大肠杆菌RosettaBlueTM中，经IPTG诱导表达。表达蛋白经Ni柱亲和层析纯化后，通过Western blot和氨基酸测序鉴定目的蛋白。
结果与结论：大肠杆菌的融合蛋白BAA表达量约占总蛋白的40%，纯化后蛋其白纯度可达90%左右。Western Blot结果显示在相对分子质量45 000处可见一特异目的条带，氨基酸测序显示N端15个氨基酸与目的蛋白完全相同。结果证实，融合蛋白BAA在大肠杆菌中得到高效的表达。

关键词: 蛔虫, 过敏原, ABA-1, 密码子优化, 组织构建

Abstract:

BACKGROUND: Serious allergic reaction could be caused by Ascaris anaphylactogen ABA-1 protein (Ascaris Body fluid allergen 1).
OBJECTIVE: To express the chimeric protein truncated from Ascais major allergen ABA-1 in Escherichia coli.
METHODS: The chimeric gene, obtained from GeneBank and Protein Database and named as BAA hereafter, was constructed by tailoring the coding fragments of Ascaris main allergen ABA-1, i.e., ABA-1B1, ABA-1A2 and ABA-1A1. The fusion gene BAA was synthesized and cloned into expression vector PET-44a, and transformed into Escherichia coli JM109 with the method of KCM .When the plasmid was confirmed by double restriction enzymatic digestive test with NdeⅠand PstⅠand DNA sequencing, it was transformed into Escherichia coli RosettaBlueTM step by step. The produced chimeric protein BAA was purified by nickel ion affinity chromatography, followed by identification by Western Blot and protein sequencing.
RESULTS AND CONCLUSION: Chimeric protein was inducibly expressed in Escherichia coli, with the production up to 40% of the total protein. Protein purity could reach about 90% after purification. Western Blot showed a specific band at 45 000, and amino acid sequencing identified that the 15 N-terminal amino acids were identical to the target protein. The chimeric protein has been efficiently expressed in Escherichia coli.

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