Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for develo...Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.展开更多
Objective: To observe the oxidative modification of high density lipoprotein (HDL) induced by cultured human arterial smooth muscle cells (SMCs). Methods: HDL cocultured with SMCs at 37℃ in 48 h was subjected, and na...Objective: To observe the oxidative modification of high density lipoprotein (HDL) induced by cultured human arterial smooth muscle cells (SMCs). Methods: HDL cocultured with SMCs at 37℃ in 48 h was subjected, and native HDL (N-HDL) served as control. Oxidative modification of HDL was identified by using agarose gel electrophoresis. Absorbances of conjugated diene (CD) and lipid hydroperoxide (LOOH) were measured with ultraviolet spectrophotometry at 234 and 560 nm respectively, and fluorescence intensity of thiobarbuturic acid reaction substance (TBARS) with fluorescence spectrophotometry at 550 nm emission wavelength with excitation at 515 nm. Results: In comparison with N-HDL, the electrophoretic mobility of SMCs-cocultured HDL was increased, and the contents of CD, LOOH and TBARS HDL were very significantly higher than those of the control HDL (P<0.01). Conclusion: Oxidative modification of HDL can be induced by human arterial SMCs.展开更多
基金This work was supported by the National Natural Sciences Founda- tion of China(NSFC, No. 39800057, No. 30200338) the National "863" High-tech Project Foundation (No. 102-10-01 -06) +1 种基金National Distinguished Youth Program of NSFC(No. 39525020) This wor
文摘Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
文摘Objective: To observe the oxidative modification of high density lipoprotein (HDL) induced by cultured human arterial smooth muscle cells (SMCs). Methods: HDL cocultured with SMCs at 37℃ in 48 h was subjected, and native HDL (N-HDL) served as control. Oxidative modification of HDL was identified by using agarose gel electrophoresis. Absorbances of conjugated diene (CD) and lipid hydroperoxide (LOOH) were measured with ultraviolet spectrophotometry at 234 and 560 nm respectively, and fluorescence intensity of thiobarbuturic acid reaction substance (TBARS) with fluorescence spectrophotometry at 550 nm emission wavelength with excitation at 515 nm. Results: In comparison with N-HDL, the electrophoretic mobility of SMCs-cocultured HDL was increased, and the contents of CD, LOOH and TBARS HDL were very significantly higher than those of the control HDL (P<0.01). Conclusion: Oxidative modification of HDL can be induced by human arterial SMCs.
