Selection And Preparation Of S Protein Diagnosis Antigen Epitopes Of SARS-Coronavirus And Characterization Of Its Immunocompetence

Objective: S protein antigen epitopes of SARS-Coronavirus were analyzed and selected, The conserved eptitopes from S protein were obtained by artificially synthesized antigen peptides and recombinant protein, respectively. The immunoreactivity and immunogenicity of the antigen proteins were analyzed. Our study can be developed into diagnosis kit for detecting early infection of SARS-CoV, ruling out unsure patients and carrying out epidemiological research.Methods: Major structural proteins of SARS-CoV were analyzed by bioinformatics tools and the B lymphocyte epitopes were predicted using multiple parameters. Two peptides of 28 amino acid from S1 and S2, and an artificial gene of 801 bp which covers the 304 to 571 amino acid were synthesized. The latter was cloned into pUCm-T vector and E.coli JM109 was transformed. The positive transformants pUCm-T/SARS-S1 were selected by colony color, and further analyzed by restriction enzyme digest, PCR amplification, DNA sequencing with common primers and BLAST analysis. The recovered DNA insert, derived from double disgested pUCm-T/SARS-Sl by BamH I and Sal I, was subcloned into pET-28b(+) and confirmed by PCR, restriction enzyme digest and DNA sequencing. The transformed E.coli ril was induced with 1 mM IPTG and the overexpressed protein rSARS-Sl was analyzed by SDS-PAGE and Western blot. The fusion protein which in the inclusion body(IBs) was purified under denaturing condition using Ni-NTA Spin Kit after the IBs was washed by three ways. The immunoreactivity of the fusion protein was assayed with sera of acute or convalescent SARS patients .Simultaneously, thepurified protein was used to immunize rabbits to obtain polyclonal antibodies. ELISA and Western blot were used to detect the specificity of the polyclonal antibodies to the synthesized antigen peptides, the recombinant SARS-S1 protein, SARS-CoV and its lysates.Results: Two antigen peptides were synthesized artificially and the relevant polyclonal antibodies were prepared (Abl and Ab2, respectively); the synthesized fragment of SARS-S1 gene (801bp in length) was inserted into pUCm-T. DNA sequencing proved that the synthesized gene was consistent with the published sequence in GenBank. The SI fragment was subcloned into pET-28b(+) and the plasmid pET-28b(+)/SARS-Sl was used to transform E.coli ril. Induced by IPTG, the recombinant protein rSARS-Sl achieved its highest production (47%) at 5h grown at 37 "C,mainly in the form of inclusion body. Its size was 32kD,as expected.The His Tag was confirmed by Western blot. Purified rSARS-Sl was obtained by washing the inclusion body and further affinity column purification with Ni-NTA Spin Kit,the purity of it was above 90% after the IBs was washed by three ways, and above 95% after purified by Ni-NTA Spin kit under denaturing conditions. ELISA data showed that rSARS-Sl could react with Abl but not Ab2; and rSARS-Sl could react with sera of acute or convalescent SARS patients but both synthesized peptides failed to react; sera derived from rSARS-Sl could react with peptide 1 and rSARS-Sl with a titer higher than 1:640.Conclusion:(1) Prokaryotic expression plasmid pET28b(+)/ SARS-S1 was constructed successfully, and a 32kD protein(rSARS-Sl) was expressed efficiently in E.coli ril;(2) rSARS-Sl could react with acute or convalescent SARS patients, but the peptide could not;(3) rSARS-Sl showed good immunogenicity and could induce the immune...