Expression,purification and immunogenicity evaluation of recombinant HPV58 virus-like particles
Objective To prepare recombinant human papillomavirus 58 L1 protein virus-like particles(HPV58 L1 VLPs)in Pichia pastoris and immunize mice with the purified product to evaluate the immunogenicity of HPV58 L1 VLPs.Methods The HPV58 L1 multi-copy expression plasmid was constructed and transformed into Pichia pastoris KM71.The highly expressed Pichia pastoris engineering strain was screened and cultured in 5 L fermenter.The expressed HPV58 L1 VLPs were isolated and purified,which were then used to immunize 80 female BALB/c mice at different doses by intraperitoneal injection.After four weeks,the immune effect was evaluated by median effective dose(ED50).Results Restriction analysis results showed that HPV58 L1 expression vectors were constructed correctly.Western blot,amino acid sequencing and trans-mission electron microscopy(TEM)results showed that HPV58 L1 protein was correctly expressed in Pichia pastoris and could self-assemble into VLPs at a diameter of about 50 nm.After purification by ion exchange and size exclusion chromatog-raphy,the HPV58 L1 VLPs reached a purity above 95％,and the ED50 in mouse model was 0.009 μg.Conclusion Recom-binant HPV58 L1 VLPs with good immunogenicity were successfully prepared by using Pichiapastoris.
Human papillomavirus(HPV)Virus-like particles(VLPs)Neutralizing antibodiesMedian effective dose(ED50)Pichia pastoris
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