[Identification of oligopeptides mimicking the virus attachment protein of hantaan virus].

OBJECTIVE: To identify and characterize the epitope associated with the virus attachment protein (VAP) of hantaan virus. METHODS: The monoclonal antibody 3G1 was used as the ligand to biospan from a phage-displayed 12-amino acid peptide library, then the positive phage clones were chosen and sequenced. The amino acid sequences of them were compared with that of hantaan virus G2 in homology. The characteristics of positive phage were studied by IFA and ELISA. A decapeptide combining to cell membrane was observed under laser scanning confocal microscope (LSCM). RESULTS: The conservative motif PX(1-2) HX(0-2) H displaying on positive clones shared homologous amino acid sequence with G2 96YPWHTAKCHY105. CONCLUSION: G2 96YPWHTAKCHY105 might play some roles in virus binding to host cell, and might be a possible key epitope of hantaan virus VAP.

[Development and application of an indirect immunofluorescence assay for the detection of serum immunoglobulin G to human herpesvirus 6].

AIM: To establish an indirect immunofluorescence assay(IFA) for detection of serum IgG antibody against human herpesvirus 6(HHV-6). METHODS: Cord blood mononuclear cells infected by HHV-6 strain CN(5) were used to prepare cell antigen smear, so as to establish an IFA and make an epidemical investigation on serum specimens of child-bearing age, women. RESULTS: The specificity of the IFA for HHV-6 was confirmed by absorption assay(test). The IFA detection showed that in serum specimens from 116 cases of child-bearing age, women, the positive rate of anti-HHV-6 IgG was 72.4% and geometric mean titer(GMT) was 1:61. The positive rate and GMT of serum anti-HHV-6 IgG had no differences between pregnant women and unpregnant women, neither among pregnant women at different pregnant stages. CONCLUSION: A specific IFA has been developed successfully for epidemical investigation of HHV-6 infection rate in child-bearing women.

[Analysis on patial amino acid sequence of a novel glycoprotein binding to antiherpes simplex virus monoclonal antibody CHA9].

AIM: To acquire the characteristic amino acid sequence of a novel glycoprotein of herpes simplex virus (HSV), so as to localize gene encoding the novel glycoprotein accurately. METHODS: A 12-mer phage peptide library was screened for 3 rounds by using biotinylated mAb CHA9 against new glycoprotein g30k of HSV-2. Positive phage clones were detected by ELISA. 10 positive clones were selected randomly for sequencing. Sequence alignment and hydrophobic cluster analysis were carried out. RESULTS: Most of the sequences of 10 positive phage clones were highly homologous with a core-PH/KHXHXGS-. Phages containing this motif could react specifically with the mAb CHA9 and not cross-react with other IgG. Hydrophobic cluster analysis showed that the peptide was likely to form an epitope. CONCLUSION: We obtained a characteristic short peptide which shows some characters of g30K amino acid sequence. It provides valuable clue for prediction of the open reading frame of g30K gene and will be useful to explore biological characteristics of the new glycoprotein.

[Purification and characterization of the fusion protein TGF-betaR II/Fc].

AIM: To express the fusion protein TGF-betaR II/Fc in large amounts by using recombinant Bac-TR II baculovirus expression system constructed by our laboratory and to purify and characterize it. Then, to verify whether the fusion protein TGF-betaR II/Fc can be able to block the biological activity of cytokine TGF-beta1. METHODS: The viral titer was determined by plaque forming test. The recombinant baculovirus was amplified by infecting sf9 cells. The fusion protein was purified by FPLC using protein G column. The purified product was analyzed by SDS-PAGE and the amount of target protein calculated by gray scanning. Western blot and sandwich ELISA were used to affirm the expression of the fusion protein. MTT colorimetry was used to test whether the fusion protein can block the inhibition effect of cytokine TGF-beta1 on the growth of L929 cells. It was to verify whether the fusion protein can reduce the fibronectin production in L929 cells accelerated by TGF-beta1 by western blot. RESULTS: The titer of recombinant Bac-TR II baculavirus in the primary culture fluid was 2x10(12) pfu/L. After electrophoresis, gray scanning analysis showed that the target protein accounted for 10 percent of the total protein. Western blot analysis and sandwich ELISA detection proved that the target protein has been expressed. The fusion protein could block the inhibitive effect of cytokine TGF-beta1 on the growth of L929 cells and fibronectin production in L929 cells. CONCLUSION: The fusion protein TGF-betaR II/Fc can inhibit the biological activity of TGF-beta1 in-vitro. This study will be helpful to the mass production of the fusion protein, and will facilitate its further use in the therapy of pulmonary fibrosis.