中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (18): 3341-3344.doi: 10.3969/j.issn.1673-8225.2012.18.025

• 移植与免疫 transplantation and Immunology • 上一篇    下一篇

抗原血清学识别重组表达克隆技术的优化*★

乔玉莉1，张  鹏1，苏家光2，陈志坚3，周素芳1，廖志红1，林文珍1   

  1. 1广西医科大学生物化学与分子生物学教研室，广西壮族自治区南宁市  530021；广西医科大学第一附属医院，2皮肤性病科，3检验科，广西壮族自治区南宁市  530021
  • 收稿日期:2011-11-21 修回日期:2012-03-02 出版日期:2012-04-29 发布日期:2012-04-29
  • 通讯作者: 林文珍，副教授，硕士生导师，广西医科大学生物化学与分子生物学教研室，广西壮族自治区南宁市 530021 linwenzhen@msn.com
  • 作者简介:乔玉莉★，女，1986年生，山东省菏泽市人，汉族，广西医科大学在读硕士，主要从事应用抗原血清学识别重组表达克隆技术筛选SLE相关抗原的研究。yuliqiaoqyl@126.com
  • 基金资助:

    广西自然科学基金资助项目(2010GXNSFA013162)。

Optimization of serological identification of antigens by recombinant expression cloning

Qiao Yu-li1, Zhang Peng1, Su Jia-guang2, Chen Zhi-jian3, Zhou Su-fang1, Liao Zhi-hong1, Lin Wen-zhen1   

  1. 1Department of Biochemistry &Molecular Biology, Guangxi Medical University, Nanning  530021, Guangxi Zhuang Autonomous Region, China; 2Department of Dermatology &Venereology; 3Department of Clinical Laboratories, First Affiliated Hospital of Guangxi Medical University, Nanning  530021, Guangxi Zhuang Autonomous Region, China
  • Received:2011-11-21 Revised:2012-03-02 Online:2012-04-29 Published:2012-04-29
  • Contact: Lin Wen-zhen , Associate professor, Master’s supervisor, Department of Biochemistry &Molecular Biology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China linwenzhen@msn.com
  • About author:Qiao Yu-li★, Studying for master’s degree, Department of Biochemistry &Molecular Biology, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China yuliqiaoqyl@126.com
  • Supported by:

    Natural Science Foundation of Guangxi, No.2010GXNSFA013162*

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摘要：

背景：抗原血清学识别重组表达克隆技术的应用有许多报道，但存在假阳性较高、稳定性欠佳等诸多问题。
目的：优化抗原血清学识别重组表达克隆技术，建立灵敏且稳定的技术体系。
方法：利用抗原血清学识别重组表达克隆技术，比较血清的处理方法、异丙基-β-D硫代半乳糖苷的加入方式、不同的封闭条件及培养基厚度对显色结果的影响，观察噬菌斑显色深浅、背景处理及假阳性的产生。
结果与结论：直接法预处理血清、异丙基-β-D硫代半乳糖苷浸泡硝酸纤维素膜并干燥结合脱脂奶粉进行封闭，显色效果最佳。此外选择适当的培养基厚度可有效消除边缘效应。提示血清预处理、异丙基-β-D硫代半乳糖苷的加入方式、不同封闭条件及培养基厚度等因素，在利用抗原血清学识别的重组表达克隆技术进行抗原筛选中起关键作用。
关键词：抗原血清学识别的重组表达克隆技术；抗原；cDNA文库；噬菌体
缩略语注释：SEREX：serological identification of antigens by recombinant expression cloning，抗原血清学识别重组表达克隆技术
doi:10.3969/j.issn.1673-8225.2012.18.025

关键词: 抗原血清学识别的重组表达克隆技术, 抗原, cDNA文库, 噬菌体

Abstract:

BACKGROUND: In recent years, there are many reports on serological identification of antigens by recombinant expression cloning (SEREX) technology. However, there are higher false positive and poor stability problems.
OBJECTIVE: To establish a sensitive and stability system by optimizing the SEREX technology.
METHODS: The SEREX technology was used to compare the serum pretreat methods, isopropyl-beta-D-thiogalactoside adding methods, and the effect of different blocking conditions and medium thickness on the results of the coloration, and the plaque stained depth or not, background clean or not and false positive produce or not were observed.   
RESULTS AND CONCLUSION: The best result was obtained using following conditions: the serum was incubated directly;  nitrocellulose membrane was immersed in isopropyl-beta-D-thiogalactoside and dried in air and then blocked with skimmed-milk; the thickness of the medium could efficiently eliminate the brim effect. It suggests that the serum pretreat methods, isopropyl-beta-D-thiogalactoside adding methods, as well as different blocking conditions and medium thickness play a key role in the selection of antigen by using SEREX technology.

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