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| Preparation and Purification of the Recombinant Protein of Venezuelan Equine Encephalitis Virus and Construction of GICA Method |
| WANG Zhen-dong1, WANG Lin-lin1,2, 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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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.
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Received: 11 January 2011
Published: 25 July 2011
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