Expression.purification and antigenicity of a recombinant respiratory syncyttal virus fusion gene encoded protein(amino acids 168-289)in the insect cells / 中华检验医学杂志

Objective To explore the antigenicity of the recombinant respiration syncytial virus (RSV)fusion protein (amino acids 168-289) encoded by 546-881 bases of the fusion gene expressed in insect baculoviruses expression system.Methods The fragment F' of fusion gene 546-881 bases was amplified from viral RNA( Long strain ) by the reverse transcription-polymerase chain reaction(RT-PCR).F' was inserted into transfer vector pBacPAK9 and the recombinant plasmid pBacRSV F' was constructed.Sf9 insect cells were then co-transfeeted with a mixture of recombinant plasmids pBacRSV F' and linearized BacPAK6 viral DNA( Bsu36 Ⅰ -digested).The recombinant baculoviruses BacPAK F' was constructed and was able to express the recombinant protein in Sf9 insect cells.The recombinant protein was purified by Ni2+ NTA chromatography and its antigenicity was identified by Western blot(WB) analysis.Specimens including the nasopharyngeal aspirates(NPAs) and sera were collected from 33 infants and young children with acute lower respiration tract infection. Indirect immunofluorecenee assay (IFA) and WB were used to detect the RSV antigen in NPAs and the anti-RSV antibody in sera respectively.Results The recombinant protein (molecular weight 13 000) was expressed in Sf9 insect cells.WB analysis demonstrated that the purified recombinant protein had a specific RSV antibody-binding activity. The recombinant protein could be recognized by positive serum infected by RSV.The positive rate was 27.3% and 33.3% respectively.There was no significant difference between them(X2 = 0.29 ,P ＞ 0.05).Conclusion The recombinant respiratory syncytial virus fusion gene (546-881 bp) encoding protein is expressed in Sf9 insect cells and it has strong antigenicity and could have clinical application value for detection of RSV infection.

Immunogenicity of a novel vaccine with VLP as vector for Wilm's tumor / 中华普通外科杂志

Objective To develop a vaccine for Wilm's tumor in order to lay a foundation for effective treatment and substitute for the radiotherapy and chemotherapy of this tumor. Methods DNA fragments encoding the WT1 antigens (including, HLA-A * 2402 and HLA-A * 2402x), and DNA fragments encoding the couple antigens HLA-A * 2402-HBc and HLA-A * 2402x-HBc were cloned and inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation and baculovirus expression pFactbac respectively, then transfected into COS-7 cells and Sf 9 cell respectively. The expression of the target proteins in the cells were identified by CPE, SDS-PAGE and electron microscopy. Virus-like particles were observed under electron microscopy. Results The fusion gene of HLA-A * 2402 and HLA-A * 2402x and HBc express virus-like particle protein. Conclusions This preliminary result shows a hopeful future in developing an effective immunotherapy to Wilm's tumor.

Structure-activity relationships of anti-HIV-1 peptides with disulfide linkage between D- and L-cysteine at positions i and i+3, respectively, derived from HIV-1 gp41 C-peptide

The constrained alpha-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an alpha-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the alpha-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less alpha-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.

Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.