| Objective Human coronavirus 229E ( HCoV-229E ) is divided into Nidovirales,Coronaviridae Coronavirus. Its genome is a single-strand positive sense RNA, and its genome all-length about 27kb. Now we have known that HCoV-229E encoded 11 varietal proteins. They have 4 constitutive proteins: spike protein, membrane protein, envelope protein and nucleocapsid protein. Spike protein trimer form spike and exsertion virus peplos on the surface. Spike protein is archi-surface antigen component of virus, it has acceptor binding and membrane fusion activity. Spike protein has important effect at tissue tropism ,cell-fusion and toxicity. Spike protein consists of S1 and S2 subunit. S1 is head and S2 is rod-shape part, intermolecul ar effort can bind each other. S1 can bind with host cell epimembranal acceptor. S2 can fix integral Spike protein on the cellular membrane and mediate cell-fusion with virus and host cell. There are significant antigen determinant of virus in Spike protein.In my research, after cloning S1 gene fragment of HCoV-229E coronavirus (1249-1642 nucleotide) and expressing S1 protein(S1 417—547 amino acids),we have purified the S1 protein.Method S1 gene fragment is synthesized and cloned to pET21a vector.After sequencing ,the S1 gene fragment was digested with Nde I and Xho I and inserted into prokaryotic expression vector pET-21a.The recombinant pET21a/ HCoV-229E S1 was transferred into an expression strain E.coli BL21(DE3) and induced by IPTG express fusion protein.The protein was identified by Western blot and purified with a kit.Results It was found that the fusion protein of 14.4kD was expressed mainly as inclusion body, The fragement of S1 protein was confirmed by Western blot.Conclusion It concludes prokaryotic expression vector pET21a/ HCoV-229E S1 is successfully constructed and S1 protein was expressed in E.coli BL21(DE3). Our work may provide foundation for further research of protein immunogenicity and the detection of anti-virus vaccines. |
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