ABSTRACT
Objective@#To prepare peptide minotope-based recombinant diagnostic antigen of Epstein-Barr virus (EBV) infection and evaluate its antigenicity preliminarily.@*Methods@#With Trx at the N-terminal and His tag at the C-terminal, the peptide minotope of EBV (GP125, F1, A2, A3C2) was expressed in Escherichia coli and purified by affinity and anion exchange chromatography (designated 'H58’); based on antigenicity of H58 identified by Western blotting (WB), we constructed and evaluated a novel early diagnostic ELISA for EBV infection.@*Results@#The soluble H58 protein with high concentration (2.8 mg/ml) and purity (99.01) was obtained; WB analysis found that there was an obvious band (28 ×103) on the NC membrane, using H58 anti-Trx monoclonal antibody or acute-phase sera of EBV infection as the first antibody. With the novel ELISA, 50 positive sera of EBV infection and 50 negative sera were detected, displaying that the grouping of OD value of positive serum (95%CI: 1.233-1.489) and negative serum (95%CI: 0.113-0.159) was different (P<0.05) with the sensitivity 98.0%, specificity 96.0% and kappa value 0.940.@*Conclusions@#By E. coli expression and affinity and ion exchange chromatography purification, the peptide minotope-based recombinant diagnostic antigen of EBV infection was obtained with excellent antigenicity, which could be applied for serological detection of EBV infection.
ABSTRACT
Objective@#To study the expression and purification of the chimeric virus-like particles displaying epitopes of EV71 as a candidate of enterovirus 71 gene recombined vaccinet.@*Methods@#The fusion protein hepatitis B core (HBc)-SP70 was constructed by inserting SP70 into the main immunogenic region of truncated hepatitis B core antigen (HBcAg) sequence, expressed in E. Coli, and purified through sonication, ion exchange chromatography, CsCl cushion centrifugation and density gradient centrifugation. Then its antigenicity was detected by ELISA and Western blot assay.@*Results@#Recombinant plasmid pHBc-SP70 was successfully constructed. And the soluble fusion protein was efficiently expressed induced by IPTG. The purity of the chimeric virus-like particles (VLPs) was up to 90% after the purification process described in method . The purified fusion protein HBc-SP70 could be spontaneously folded and assembled into empty virus-like particles and react with the monoclonal antibodies against HBc and SP70.@*Conclusions@#The chimeric VLPs displaying epitopes of EV71 were efficiently expressed and purified in E. Coli. with excellent antigenicity, which laid a foundation for evaluation of the immune effect evoked by this EV71 gene recombined vaccine.
ABSTRACT
Objective To clone,express and purify thioredoxin (named as N5),a specific diagnostic antigen of hepatitis E virus (HEV),and to initially evaluate its antigenicity and serological test performance.Methods Based on the gene sequences of HEV-ORF2 and carboxyl terminal ORF3 on GenBank,the codon was optimized by the Escherichia coli codon preference,inserted it into prokaryotic expression vector M48 following total gene synthesization,and expressed in Escherichia coli fusion protein N5 recombined with Thioredoxin (TRX).Fusion protein was purified in affinity chromatography,evaluating its antigenicity with Western blot technology,then evaluating its serological test performance using the negative and positive serum samples confirmed of HEV infection with laboratory and clinical tests.Results The recombinant plasmid expressing N5 diagnostic antigen was successfully established; high-level expression and purification to obtain soluble diagnostic antigens; Western blot results indicating fusion protein N5 can be bound specifically with the serum of HEV IgM antibody positive,showing satisfactory antigencity; using fusion protein N5 as the capture antigen to build indirect ELISA,testing 40 serum samples of HEV cases confirmed by pathogen detection and clinical diagnosis and 40 serum samples of healthy people,with the sensitivity and specificity of 95％ (38/40) and 90％ (36/40) respectively.Conclusion Recombinant plasmid expressing the HEV diagnostic antigen recombined with thioredoxin was successfully established,and soluble fusion protein N5 was obtained with high expression and strong antigenicity,promising in its future applications.