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Expression of Chimeric Protein of Norovirus P Domain and Neutralizing Epitopes of EV71 in Prokaryotic Escherichia coli

LI Chao;LIU Bo;TAO Yu-fen;LI Xin-tong;LIU Jian-sheng;LIU Hong-qi;Infection and Immunity Laboratory,Institute of Medical Biology,Chinese Academy of Medical Sciences &Peking Union Medical College;  
Objective: To construct recombinant plasmids and express chimeric proteins of neutralizing epitopes of Human Enterovirus 71 and Norovirus P domain. Methods: Nucleotide sequences of the neutralizing epitopes of EV71 were designed and optimized according to their reported sequences of amino acids and the codon bias usage of E. coli for expression. These nucleotide fragments were cloned into the plasmid vector containing norovirus P domain and GST tag via the loop2 of P domain,which was confirmed by sequencing. The constructs were transformed into the bacteria BL21( DE3) and induced with IPTG for protein expression. The GST-fused proteins were purified via GST affinity beads. Immunoblot analysis was used to detect the GST tag and evaluate the immunogenicity of chimeric proteins. Results: Seven recombinant plasmids were constructed successfully as confirmed by sequencing. The expressed 7 chimeric proteins were expressed and soluble in E. coli,which was determined by SDS-PAGE and Commassie blue staining. Immunoblot analysis via the anti-GST antibody revealed the expression of GST-fused proteins. Further analysis of immunogenicity showed that all 7 chimeric proteins could react with anti-No V P domain antibody. Five out of 7 chimeric proteins could recognize anti-EV71 polyclonal antibody except two epitopes of SP55 and SP28 chimeric proteins. Conclusion: The chimeric proteins of No V P domain and EV71 neutralizing epitopes were successfully expressed and antigenic,which lays a solid foundation for developing the bivalent subunit vaccine and the detection kit for No V and EV71 viruses.
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