Objective To synthesize an inhibitory peptide against serine protease of hepatitis C virus(HCV)and explore its inhibition ability in vitro. Methods Based on the sequence characteristics of four natural substrates of protease in HCV,the gene sequence of an inhibitory peptide against HCV serine protease was designed and directly synthesized by PCR. The segment was subcloned into prokaryotic expression vector pBVIL1,resulting in the construction of the recombinant plasmid pBVIL1/IP,which was then transformed into E. coli HB101 strain. Purified by ion exchange chromatography,the expressed protein was added into an in vitro system,which was comprised of the inhibitory peptide(expressed protein),protease and the substrate,i.e. a NS5A-B fragment,in phosphate buffer. Then SDS-PAGE was performed to test the inhibition effect of the polypeptide. Results The recombinant expression vector pBVIL1/IP containing target gene was successfully constructed. The peptide expressed as inclusion boay was identified by SDS-PAGE. The degradation of protease substrate NS5A-B fragment is inhibited proportionally with the increasing concentrations of the peptide. Conclusion The recombinant peptide shows inhibitory effect on HCV protease in vitro.

Objective To investigate immune responses and protective effect induced by two recombinant proteins of hepatitis C virus(HCV)in BALB/c mice.Methods BALB/c mice were immunized with recombinant proteins HCV-T and(or)HCV-F4HVR1 three times.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Five mice were sacrificed after 14 days of the last immunization.Splenic cells were isolated and levels of interferon(IFN)-γ,interleukin(IL)-4 and cytotoxic T lymphocyte(CTL)cytotoxicity assay were measured in vitro.The remaining mice were subcutaneously injected with 1.0×106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results Compared with phosphate bufter saline(PBS)group,combined immunization with HCV-T and HCV-F4HVR1 induced higher levels of specific IgG against HCV-F4HVR1(t=3.815,3.762,P＜0.05),HCV-NS3-specific CTL response(t=3.971,P＜0.05)and IL-4(t=3.512,3.417,P＜0.05)and IFN-γ(t=3.813,3.426,3.671,P＜0.05)secretions.Conclusion High levels of specific humoral immunity and cellular immunity are induced in vivo after combined immunization with HCV-T and HCV-F4 HVR1,which could effectively prevent from the attack of SP2/0-NS3 cells.

Objective To establish a sandwich enzyme-linked immunosorbent (ELISA) method for early diagnosis of hepatitis C. Methods Immunization of Balb/c mice with hepatitis C core antigen were prepared by genetic engineering. Mouse monoclonal antibodies (McAb) to anti-HCV-core Ag were obtained. A sandwich ELISA kit for detecting HCV-core Ag was developed by using four strains of anti-HCV-core Ag McAb. One hundred and twenty five serum specimens with increased ALT but negative for anti-HCV, anti-HIV, and anti-Tp tests were tested. Results Nine of the 125 specimens were positive for HCV-core Ag. Conclusion The double sandwich ELISA kits for detecting HCV-core Ag may be useful for the early diagnosis of hepatitis C .

Objective To investigate the cellular and humoral immune responses and protective effect induced by co-immunization with two multi-epitope combinant antigens. Methods Mice were co-im-munized with the muhi-epitope HCV-T and HCV-E1 antigens three times. Sera antibodies IgG, IgG1 and IgG2a were tested by ELISA. Spleens from BALB/c mice immunized were removed 10 days after the last im-munization. CTL activity was assessed using LDH cytotoxicity assay kit. IFN-γ- and IL-4-secreting cells were quantified using ELISPOT kit. Two weeks after the final immunization, the mice were challenged sub-cutaneously(s, c. ) at the back with 106 SP2/0-NS3 cells, and protective effect was observed. For therapy, 106 SP2/0-NS3 cells were implanted into the back of BALB/c mice. Seven days later, mice were immuniza-tion three times. Therapy effect was observed. Results Co-immunization with HCV-T and HCV-E1 induced high tiers of HCV-El-specific IgG, IgG1 and IgG2a antibodies, and high level of CTL activity. Synergistic effect in frequencies of both specific IFN-γ-secreting cells and IL-4-secreting cells was observed in mice co-immunized. Prophylactic as well as therapeutic administration of mT + mE1 in mice led to protecting mice against SP2/0-NS3 cells. These results suggested that mT + mE1 was potential as a prophylactic as well as therapeutic HCV vaccine. Conclusion Co-immunization with HCV-T + HCV-EI induced protective humor-al and cellular immune response. HCV-T + HCV-E1 was potential as a recombinant HCV vaccine.

Objective To design a complex hepatitis C vires(HCV)=E1 antigen,and to search its application in HCV vaccine and diagnosis test.Methods Through consulting the database and widely comparison of sequences from HCV E1 of different genotype,the representative immunodominant epitope sequences were selected from all the six genotypes.Their genes were deduced according to the preference codon in E.coli and three fragments were designed to contain the six epitopes.They were chemically synthesized and cloned into pBVIL1 vector separately.The cloned fragments were conjugated together profiting from pBVIL1's property,and then a doubled pan-DR helper T cell epitopes(PADRE)gene was inserted to form a complex expression plasmid.The transformed E.coli cells with this plasmid were cultured and induced to express recombinant protein and the antigenic activity of the product was tested.Results An expressing plasmid containing 8 epitopes from HCV genotype 1a,1b,2a,3a,4a,6a and a doubled PADRE sequences was constructed successfully and the engineering E.coli transformed with this plasmid highly expressed after inducing culture at 42℃ in an inclusion manner.The immunological activity of the purified recombinsnt multi-epitope antigen shows that:(1)It can react with a great part of sera from HCV positive patients by indirect ELISA.(2)It can induce notable specific humoral immunity in injected mice.Conclusion The novel constructed expressing plasmid and its product may be useful in study of a newly HCV vaccine as well as an antigen for HCV immunoassays.

Objective To research CpG and Al(OH)3 adjuvants enhancing immunogenicity of hepatitis C virus(HCV) recombinant ptotein combined vaccine(TIE).Methods BALB/c mice were immunized with candidate vaccine TFE using CpG,Al(OH)3,Al(OH) 3 + CpG,or freund's adjuvant(FA) as the adjuvant.Five mice were sacrificed after 10 d of the last immunization.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Splenic cells were isolated and levels of IFN-γ,IL-4 and cytotoxic T lymphocyte(CTL) cytotoxicity assay were messuredin vitro.The remaining mice were subcutaneouly injected with 1 × 106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results The specific CTL activity of TFE + A1(OH) 3 + CpG group was higher than TFE + FA group and TFE + CpG group(P ＜ 0.05).The level of IFN-γsecreting cells in TFE + Al(OH)3 + CpG group was higher than that in TFE + M(OH)3 group or TFE + CpG group(P ＜ 0.05).Conclusion Combining Al(OH) 3 and CpG could enhance specific cellular immunogenicity of candidate HCV vaccine TFE.TFE + M(OH) 3 + CpG could effetively prevent the attack of tumor cell SP2/0-NS3 expressing nonstructural protein NS3 of HCV.

Objective To investigate the epidemic of severe acute respiratory symdrome(SARS) in Beijing blood donors and make guidance for assuring blood safety during SARS epidemic.Methods Using SARSCoV Ab ELISA Kits, specimens from 2357 donors from Beijing during SARS epidemic phase,1079 preserved samples from Beijing donors collected well before the SARS epidemic,1183 donors from Shandong and Hunan provinces where no SARS had been reported were screed for IgG,IgM,and total antibodies against SARS coronavirus.Donors with reactive samples were followed up,RT PCR were performed to detect the SARS CoV RNA.Results There was no significant difference between the 3 groups of specimens and there was no SARS epidemic or subclinical SARS infections among Beijing blood donors.Conclusion Instead of blood SARS CoV Ab screening, we should focus on the donors inquiry,physical examination and education to prevent SARS transmission by transfusion.