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.

Objective In order to further accurate diagnosis of the special serum with HDV IgM antibody positive but conventional HBV five detection negative.Methods designing the specific primers,by extracting the nucleic acid of the virus in the serum,amplifying the virus genome conservative region,and then blast the sequences in Genebak.Results In this study,the HDV and HBV conserved regions were amplified in the serum,after blast,the amplified sequences were found to be consistent with the conserved regions of HBV and HDV.Conclusion In clinical,HDV infection will appears in the serum of HDV antibody IgM positive but HBV S antigen negative,it should cause more attention in clinic for HDV infected.

Objective:A subunit vaccine against SARS-CoV-2 BA.2 variant was prepared by prokaryotic expression system and its immunogenicity was evaluated.Methods:The recombinant plasmid of BA.2 variant RBD and the tandem of RBD and TT-P2 epitopes was constructed by molecular cloning technology, and recombinant proteins So and Sot were obtained by protein purification technology. Mice were immunized by intramuscular injection after mixing protein So/Sot as immunogens with Al(OH) 3 adjuvant to evaluate the effect of cellular and humoral immunity. Results:High purity soluble protein were obtained by dialysis and renaturation after expressed by prokaryotic system. The number of antigen-specific T lymphocytes secreting IFN-γ and IL-4 (213.7±0.6 and 311.7±1.5) in the Sot group induced by mice was significantly higher than that in the So group (94.3±16.8 and 185.7±4.2) ( P<0.001). The serum antibody level induced by Sot group was higher than that in the So group, the geometric mean titer (GMT) of neutralizing antibodies against BA.2 strain were 588 and 337, respectively ( P<0.05). Sot protein induced Th1/Th2 mixed type immune response with the predominance of Th2 type. Conclusions:The protein subunit vaccine expressed by the prokaryotic system have excellent cellular and humoral immunogenicity, which provides a strong theoretical basis for the development of the protein subunit vaccines of the SARS-CoV-2 Omicron variant.

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.

Objective:To analyze the genotype distribution of acute hepatitis B virus in China.Methods:A total of six hundred and twenty acute Hepatitis B cases reported to China Information System for Diseases Control and Prevention from 2015 to 2017 were selected. First, the full-length HBV genome was obtained by nested PCR amplification. In addition, the HBV genotype was determined by constructing a phylogeny tree. Finally, using primarydata, HBV genotype distribution was analyzed.Results:A total of 519 (83.71%, 519/620) sequences were obtained genotype of 620 acute hepatitis B cases, including A (0.19%, 1/519), B (27.17%, 141/519), C (62.04%, 322/519), D (9.06%, 47/519), I (0.77%, 4/519) and C/D (0.77%, 4/519); B2(95.03%, 134/141) and C2 (72.67%, 234/322) were the two major subgenotypes. Genotypes were distributed differently in seven regions of China. The proportion of genotype C appeared higher in Northeast China (94.55%, 52/55), North China (93.85%, 61/65), East China (78.87%, 56/71), and South China (58.14%, 50/86). The proportion of genotype B was higher in Central China (58.07%, 36/62) and Southwest China (52.94%, 45/85), the proportion of genotype D was the highest in Northwest China (48.42%, 46/95). A total of 515 cases were classified as serotypes, including 'adr' (57.48%, 296/515), 'adw' (30.87%, 159/515), 'ayr' (0.19%, 1/515), and 'ayw' (11.46%, 59/515). Genotype B was dominated by 'adw' serotype (92.14%, 129/140), genotype C was dominated by 'adr' serotype (91.88%, 294/320),all genotype D were 'ayw' serotype. The genotype of acute hepatitis B was correlated with serotype, 'adw' was dominant in genotype B, 'adr' was dominant in genotype C and 'ayw' was dominant in genotype D.In different gender and age group, there was no statistical significance ingenotype distribution ( P>0.05). Conclusions:The genotype of acute hepatitis B in China from 2015 to 2017 was mainly B, C, and D; genotype C was dominant in the Northeast China,North China, East China and South China; B and C were common in Central and Southwest China, and genotype B was dominant. Genotype D was primarily distributed in Northwest China. The genotype of acute hepatitis B was correlated with serotype, 'adw' was dominant in genotype B, 'adr' was dominant in genotype C and 'ayw' was dominant in genotype D. There was no difference in the distribution of acute hepatitis B genotypes among different genders and age groups.

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.