委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立

中国生物工程杂志

2011, Vol. 31

Issue (7): 104-108

技术与方法

委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立

王振东¹, 王林林^1,2, 杨宇², 杨永莉², 王静²

1. 沈阳农业大学生物科学技术学院沈阳 110866;
2. 中国检验检疫科学研究院北京 100123

Preparation and Purification of the Recombinant Protein of Venezuelan Equine Encephalitis Virus and Construction of GICA Method

WANG Zhen-dong¹, WANG Lin-lin^1,2, YANG Yu², YANG Yong-li², WANG Jing²

1. Shenyang Agricultural University, College of Bioscience and Biotechnology,Shenyang 110866,China;
2. Chinese Academy of Inspection and Quarantine, Beijing 100123, China

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摘要：

目的:表达委内瑞拉马脑炎病毒E2重组蛋白,并结合胶体金免疫层析技术建立一种简便检测委内瑞拉马脑炎病毒特异性抗体的方法。方法:利用已经构建的表达E2抗原的工程菌, 用IPTG诱导, 表达蛋白主要以包涵体的形式存在。通过一系列条件的变性、复性、透析,所制抗原用以包被硝酸纤维素膜,利用胶体金标记和免疫层析技术,建立委内瑞拉马脑炎病毒快速检测方法。对该方法的敏感性、特异性和稳定性作出评价。结果:重组工程菌可表达分子质量为 40 kDa的目的蛋白, 纯化后的蛋白质经SDS-PAGE显示纯度达95%以上。建立的检测方法可在20 min内完成检测。对症状相似及近缘的其他病毒进行检测,均无非特异反应。试纸条在37℃下保存2周,检测结果不变。该方法与R&D公司商品化的ELISA试剂盒灵敏度检测无明显差异;对92份阴性血清进行检测,两种检测方法的符合率为96.7%。结论:重组委内瑞拉马脑炎病毒蛋白产生的包涵体变性复性后生具有良好的重复性和稳定性, 可作为委内瑞拉马脑炎病毒多种检测方法的抗原原料。胶体金免疫层析法具有快速、灵敏、特异、稳定的特点,适用于现场检测。

关键词： 包涵体; 蛋白质纯化; 委内瑞拉马脑炎病毒; 胶体金; 免疫层析技术

Abstract:

Objective: To establish a colloidal gold immunochromatography assay for rapid quantitative detection of specific antibody to Venezuelan Equine Encephalitis Virus(VEEV) using the expressed E2 recombination protein. Method: The expressed E2 recombination protein was expressed in the form of inclusion body which used Isopropyl-β-D-thiogalactopyranoside (IPTG) as revulsant. The expressed inclusion body was denatured, renatured and dialyzed. With colloidal gold labeling Staphylococcal protein A and immune-chromatography technics, The colloidal gold immunochromatography assay method(GICA) was prepared and applied to detect fast the VEEV serum. The VEEV antigen was coated on the test region. This study evaluates the sensitivity, specificity and stability of this method. Results: SDS-PAGE revealed the VEEV expression product had a 40 kDa protein, as expected. And the purity of the optimized inclusion body was above 95%.The detection can be completed within 20min with this method with sensitivity of the established method is the same as R&D the antibody of VEEV ELISA. The other homologous virus and the viruses which had the similar symptom had nonspecific reaction. The test strip could save two weeks in 37℃ with no changed results. Ninety-two blood serum samples were tested by means of gold-immunochromatography and ELISA, the rate of coincidence is 96. 7%, when compared with that of ELISA. Conclusion: The inclusion body of the VEEV can be significantly and stably improved after denaturation and renaturation. So the inclusion body can be used as a candidate for many testing methods. The GICA can detect the antibody of VEEV fast, sensitive, specific and stably in samples, and suitable for field detection.

Key words: Inclusion body Purification of protein Venezuelan equine encephalitis virus Colloidal gold Immunechromatography

收稿日期: 2011-01-11 出版日期: 2011-07-25

ZTFLH:

Q789

基金资助:

国家自然科学基金资助项目(20672088)

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引用本文:

王振东, 王林林, 杨宇, 杨永莉, 王静. 委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立[J]. 中国生物工程杂志, 2011, 31(7): 104-108.

WANG Zhen-dong, WANG Lin-lin, YANG Yu, YANG Yong-li, WANG Jing. Preparation and Purification of the Recombinant Protein of Venezuelan Equine Encephalitis Virus and Construction of GICA Method. China Biotechnology, 2011, 31(7): 104-108.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I7/104

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