Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rou...Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.展开更多
The purpose of the study was to screen the antigenic epitope with monoclonal antibody against hepatitis G virus envelope protein 2 (HGV E2) from phage-displayed constrained nonapeptide library (PVⅢ9aa-cys). E. coli...The purpose of the study was to screen the antigenic epitope with monoclonal antibody against hepatitis G virus envelope protein 2 (HGV E2) from phage-displayed constrained nonapeptide library (PVⅢ9aa-cys). E. coli XLl-blue infected with PVⅢ9aa-cys was spread on 2×YT plates containing ampicillin and tetracycline. Transducting unit(TU) of PVⅢ9aa-cys was about 1.6×10 12 /ml by calculating the number of clones. The DNA sequence of PVⅢ9aa-cys, determined with cycle sequencing, was found randomly arranged in NNN order. Both ends of the nonapeptide(NNN) 9 linked with a cysteine respectively. Through four rounds biopanning of PVⅢ9aa-cys with MAb M-13-IgG, 6 of 14 positive clones were proved sharing the consensus aa sequence VXXSPL while 4 clones shared sequence VXSPL. Sequence VX(X) SPL was of homology with VRSPL of HGV E2 between aa 218-222. Average 0.D. value(A 450 ) of 10 phage clones with the consensus aa sequence were higher than those of the other 4 clones and PVⅢ9aa-cys(P<0.05). These results demonstrate the possibility that sequence VRSPL is an antigenic epitope of HGV E2. This work provides new information for the diagnosis of HGV infection as well as vaccine development of HGV.展开更多
文摘Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.
文摘The purpose of the study was to screen the antigenic epitope with monoclonal antibody against hepatitis G virus envelope protein 2 (HGV E2) from phage-displayed constrained nonapeptide library (PVⅢ9aa-cys). E. coli XLl-blue infected with PVⅢ9aa-cys was spread on 2×YT plates containing ampicillin and tetracycline. Transducting unit(TU) of PVⅢ9aa-cys was about 1.6×10 12 /ml by calculating the number of clones. The DNA sequence of PVⅢ9aa-cys, determined with cycle sequencing, was found randomly arranged in NNN order. Both ends of the nonapeptide(NNN) 9 linked with a cysteine respectively. Through four rounds biopanning of PVⅢ9aa-cys with MAb M-13-IgG, 6 of 14 positive clones were proved sharing the consensus aa sequence VXXSPL while 4 clones shared sequence VXSPL. Sequence VX(X) SPL was of homology with VRSPL of HGV E2 between aa 218-222. Average 0.D. value(A 450 ) of 10 phage clones with the consensus aa sequence were higher than those of the other 4 clones and PVⅢ9aa-cys(P<0.05). These results demonstrate the possibility that sequence VRSPL is an antigenic epitope of HGV E2. This work provides new information for the diagnosis of HGV infection as well as vaccine development of HGV.
