To screen and characterize human phage antibody to hepatitis C virus E2 antigen. The recombinant phages were panned by HCV E2 antigen which was coated in a microwell plate. After three rounds of biopanning, 56 clones specific to HCV E2 antigen were obtained. The specificity of scFv was determined by ELISA method and cross reaction with BSA and competitive inhibition assay. HCV E2 phage antibody had a specific combination character with recombinant hepatitis C virus E2 antigen. The DNA sequence data showed that the scFv coding gene was 771bp in size

To screen anti idiotypic single chain variable fragments(anti Id scFv)against hepatitis C virus NS4A(HCV NS4A)so as to lay a foundation for developing anti Id scFv vaccine againist the hepatitis C virus. The recombinant phage antibody library was panned by hepatitis C virus NS4A monoclonal antibody which was coated in a microwell plate. After five rounds of biopanning, 82 clones specific to HCV NS4A antibody were determined with the enzyme linked immunoadsorbent assay(ELISA). The specificity of anti idiotypic scFv was identified by ELISA and competitive inhibition assay. The DNA sequence of the positive clone was determined. The result showed that HCV NS4A anti Id scFv had a specific combination character with hepatitis C virus NS4A monoclonal antibody. The DNA sequence data showed that the anti Id scFv coding gene included 789 bp. The results suggested that the anti Id scFv fragments to HCV NS4A monoclonal antibody could be successfully selected by recombinant phage antibody library screening technique, which might pave a way for the study of new preventive and therapeutic strategy of hepatitis C using anti Id scFv.

To screen HCV core mimotopes,HCV core monoclonal antibody was used as immobilized molecule,and a 12 mer phage peptide library was biopanned and positive clones were selected by enzyme linked immunoadsorbent assay(ELISA), competitive inhibition assay, and DNA sequencing. 10 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results,one epitope was confirmed as a mimotope of HCV core protein. The study might provide a new approach for HCV therapy and vaccine development.

Objective To screen HCV NS3 mimotopes by using monoclonal antibody and phage peptide library. Methods Using HCV NS3 monoclonal antibody as selective molecule, a 7 mer phage peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. Results 13 positive clones were chosen for DNA sequencing. From the experiment and sequen-cing comparsion results, one epitope was confirmed as the mimotope of HCV NS3. Conclusions HCV mimotope is obtained by phage peptide library screening.

Objective To screen the HCV NS4A binding protein. Methods By using HCV NS4A as a solidified selective molecule, the T7 select human liver cDNA library was biopanned and the positive clones were selected. After screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Two positive plaques were chosen for DNA sequencing. Results The binding protein of HCV NS4A was identified as mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MAPKAPK5) by BLAST. Conclusion This approach provides a new way for the study of the pathogenic mechanism of HCV infection.

Objective To screen the HBV core promoter binding protein, and to investigate their potential role in the replication of HBV DNA. Methods By using HBV core promoter being used as a selective molecule, the T7select human liver cDNA library was biopanned and the positive clones were selected. Results After phage display screening, the positive plaques was amplified and then cloned into the pGEM-Teasy vector. Six positive plaques were chosen for DNA sequencing. The binding protein of HBV core promoter was identified as caboxypeptidase N(CPN) by BLAST. Conclusion The results suggest that phage display screening of binding protein of HBV core protein provides a new approach to study the replication mechanism of HBV DNA.

Objective To express soluble human anti-idiotypic single chain Fv to hepatitis C core protein in E.coli. Methods Using phage display technique, the semisynthetic phage library was panned by HCV core monoclonal antibody which was coated in a microtiter plate. After three rounds of biopanning, 53 clones were identified specific to HCV core antibody. The specificity of anti-idiotypic scFv was determined by ELISA. After digested with Sfi/Not, the selected HCV core anti-idiotypic scFv positive clone was subcloned into the vector pCANTAB5E for the expression of E-tagged soluble anti-idiotypic scFv. The E.coli XL1-Blue was transformed and induced by IPTG. The specificity of anti-Id scFv was evaluated with ELISA. Results HCV core anti-Id scFv DNA digestion and sequence data showed that the scFv gene was composed of 774bp. ELISA results demonstrated that the soluble human HCV core anti-idiotypic scFv to HCV core monoclonal antibody had a specific combination character. The molecular weight of expressed HCV core anti-idiotypic scFv was 28kD as shown by SDS-PAGE. Conclusion HCV core anti-Id scFv has been successfully expressed in E.coli.

Objective To detect hepatitis C virus core antigen in 7721 cells transfected with HCV cDNA by immunohistochemistry method with human single chain Fv antibody(scFv). Methods The recombinant phages were panned by core antigen which was coated in a microtiter plate. After three rounds of biopanning, 48 clones were identified specific to core antigen. The affinity and specificity of scFv were evaluated by ELISA and immunohistochemistry. Results ScFv-core DNA digestion and sequence data showed that the scFv gene was composed of 774bp. Conclusion Human single chain Fv antibody against HCV core antigen has a specific combining capacity with hepatitis C virus core antigen.

Objective To screen promoter DNA-binding protein of inducible nitric oxide synthase gene by using phage display technique from human liver cDNA library, and to study the expression and regulation mechanism of iNOS gene. Methods The sequence of iNOS promoter was identified in GenBank by bioinformatics based on the open reading frame(ORF) of iNOS gene and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was subcloned into pCAT3-Basic reporter vector, named as pCAT3-iNOSp. The HepG2 cell line was then transfected with pCAT3-Basic to serve as negative control, and pCAT3-promoter which contains the promoter region of CMV served as the positive control subject, and pCAT3-iNOSp served as the test subject, respectively. The choloraphenical acetyltransferase(CAT)activity was determined by enzyme linked immunosorbent assay(ELISA) kit. The T7 Select human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaque was performed to amplify and PCR products were sequenced. Results The expression of CAT in transfection of pCAT3-PS1TP1p was 4.2 times as higher as pCAT3-Basic plasmid. Sequence analysis was performed in 12 positive plaque, which were the iNOSp binding protein. Conclusion The iNOS gene promoter identified in this study has shown to have transcription activity,and iNOS promoter DNA-binding proteins havs been screened. The results will be useful for further study of the expression and regulation mechanism of iNOS in liver cell.

Objective To clone amastin coding genes from different strains of Leishmania parasites. Methods Using amastin cDNA sequence as the reference, dbEST data base established by National Center Biotechnology Information (NCBI), USA, was searched by BLAST tool. A 309 bp DNA fragment of Leishmania major was found and used as the probe for the screening of a DNA library. The amastin gene of Leishmania major Abdou was cloned and sequenced. Specific primers were designed and amastin genes for Leishmania mexicana WR972, Leishmania brizeliensis and Leishmania amazonensis joseph were amplified by polymerase chain reaction. Results The amastin genes from 4 strains of Leishmania parasites were cloned and sequenced. It was found that all 4 amastin genes contained unique open reading frame of 552 bp and encoded amastin protein of 183 amino acid residues. Conclusion The amastin genes of 4 strains of Leishmania parasites were successfully cloned.