Expression And Purification Of Fusion Protein Of Human Respiratory Syncytial Virus Expressed In Eukaryotic Expression Vector

Objective:Human Respiratory syncytial virus (RSV), the widely spread virus around the world, is the most important cause of viral lower respiratory tract illness in infants and children. So far no effective vaccine is available for prophylaxis. The World Health Organization has affirmed RSV vaccines as one of the highest priority vaccine development projects. Among the encoded 11 viral proteins by RSV genome, fusion protein (F) is a membrane glycoprotein that is located in the viral envelope and mediates fusion of the virion with the target cell and the formation of syncytia. F protein owns highly conserved amino-acid identity between different group's virus, which is the most important target antigen in vaccine project, elicits cross-protective immunity and also virus-specific cytotoxic T lymphocytes (CTL). In this research, the constructed eukaryotic expression vector of pcDNA3.1(+)/F-His was transfected into COS-7 cells, and then purified by Ni+-affinity chromatograph,in order to obtain purified F protein applicable to diagnosis and vaccine, etc.Methods: F gene with 3′end thrombin cleavage site and six histidine tags was obtained by PCR. The resultant F-His gene was subcloned into pGEM-T easy vector. After confirmed by nucleotide sequence analysis, F-His gene was cloned into eukaryotic expression vector pcDNA3.1(+). The resulting pcDNA3.1(+)/F-His was transfected into COS-7 cells by Lipofectamine 2000. The transcription of F gene and the expressed protein were detected by RT-PCR, Western blot assay and indirect immunofluorescence, respectively. The purified F-His protein by Ni+-affinity chromatograph was analyzed by high performance capillary electrophoresis (HPCE).Result: DNA sequencing displayed no nonsense mutation in amplified F-His gene. The resulting recombinant plasmid pcDNA3.1(+)/F-His was confirmed by restriction endonuclease assay. The specific expression F-His protein in COS-7 cells was identified by RT-PCR, Western blot assay and indirect immunofluorescence. The recovery rate of the purified F-His protein was about 14.1% with Ni+-affinity chromatograph, and the purity was more than 99% analyzed by high performance capillary electrophoresis (HPCE).Conclusion: The established method can be used to prepare and purify RSV F protein for further investigation on monoclonal antibody and diagnosis kit, etc.