Prokaryotic expression and preparation of GST/BFRF3 fusion protein of Epstein-Barr virus for screening nasopharyngeal carcinomas |
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Authors: | HU Bo CHEN Zhong-cheng WANG Dong-ning LI Lin |
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Institution: | 1.Department of Laboratory Medicine, 2Department of Hematology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China. |
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Abstract: | AIM: To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsid antigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS: DNA extracted from the B95-8 cells was used as the templates. Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene, and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The expression of GST/BFRF3 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified by glutathione-sepharose beads. The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS: The GST/BFRF3 fusion protein was successfully expressed in E. coli. The molecular weight of the product was approximately 44 kD. The recombinant fusion protein GST/BFRF3 showed good immunoreactivity. A novel ELISA was established using GST/BFRF3 protein. Serum samples collected from the NPC patients and healthy controls were tested by this ELISA. The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65% and 87%, respectively.CONCLUSION: The recombinant protein GST/BFRF3 is expressed in E.coli, and it has diagnostic value for screening of NPC patients. |
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Keywords: | Nasopharyngeal neoplasms Epstein-Barr viral capsid antigen Prokaryotic expression |
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