ABSTRACT
Objective The specific binding peptide of Mycobacterium tuberculosis PPE17 protein was screened by phage display technique. Methods PPE17 gene was amplified from Mycobacterium tuberculosis genome, cloned into pET28a, expressed in E. coli BL21, purified by Ni2+ column, and identified by SDS-PAGE and Western blot. The purified PPE17 protein was coated into an ELISA plate and screened by phage 7 peptide library. After three rounds of panning, phage plaques were randomly selected for sequencing. DNAMAN was used to analyze and compare the amino acid sequences of the polypeptide encoded by the positive clones. Results PPE17 gene was successfully constructed and expressed, and soluble protein with molecular weight of about 37kD was obtained. From the third round of eluents, 20 plaque were randomly selected. The sequencing results could be translated into 8 polypeptide molecules, among which the polypeptide sequence repeated for 6 times was LKWGHVY. Conclusion The specific binding peptide of PPE17 protein is screened by phage display technology, which is expected to be a small molecular diagnostic reagent for the identification of this antigen.
ABSTRACT
Objective T9 provide candidate molecules for developing recombinant anti-idiotypic antibody (anti-Id) vaccine of gastric carcinoma by selection of recombinant anti-Id to monoclonal antibody ( McAb) MGb1 directed against the cancer with phage display technique.Methods Balb/c mice were immunized with MGb1 and the mRNA was isolated from the spleens of the immunized mice. The VL and VH cDNAs of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFv library. After four rounds of biopanning to the library with MGb1, the MGb1-positive clones were selected from the enriched phages by ELISA. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. Results The VL and VH cDNAs was about 320 bp and 340 bp, respectively. The ScFv DNA were about 750 bp. After four rounds panning to the phage antibody ScFv library with MGb1, 18 MGb1-positive phage clones displayed anti-Id ScFv were selected from 50 pre-selected phage clones, among which 4 clones displayed β or γ type anti-Id ScFv. Conclusion The phagedisplayed anti-Id ScFvs to McAb MGb1 are successfully selected by recombinant phage antibody technique, which might lay a foundation for screening the anti-Id ScFv possessing the characteristics of inducing anti-gastric carcinoma immunity.
ABSTRACT
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