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
OBJECTIVE: To detect the ability of polysaccharide nucleic acid fraction of bacillus calmette guerin(BCG-PSN) in inducing mIFN? in mouse. METHODS: The Balb/c mice were treated with saline, BCG-PSN and lipopolysaccharide(LPS) respectively, and mIFN? cDNA was amplified by RT-PCR. The ability of BCG-PSN in inducing mIFN? in mouse was identified through RT-PCR product with ?-actin as control. RESULTS: In normal sodium group, only ?-actin but not the mIFN? cDNA was detected, but in groups treated with BCG-PSN or LPS, mIFN? and ?-actin cDNA were all detected in RT-PCR amplification products. As compared with LPS group, there was stronger expression of mIFN? in BCG-PSN group (P
ABSTRACT
OBJECTIVE:To prepare PTD modified mice interferon gamma(PTD-mIFN-?) in order to prepare for its function research.METHODS:The code of PTD-mIFN-? fragment was amplified by PCR for 3 times,using pUC19/mIFN-? as template.The PCR product was cloned into pUC19 plasmid and then constructed into expression plasmid vector pQE80L after sequencing.After double digestion with BamH Ⅰ and Hind Ⅲ,the recombinant vector was identified in 1.2% agarose gel.The target protein and PTD-mIFN-? was checked by SDS-PAGE,Western blot method and ELISA assay,respectively.Recombinant protein was purified by nikel ion-triglycollamic acid(Ni2+-NTA) Agarose affinity chromatogram.RESULTS:The cDNA fragment encoding PTD-mIFN-? amplified by PCR was obtained.The high-level expression of recombinant protein was noted in SDS-PAGE and Western blot assay.The target protein was obtained by ELISA assay.CONCLUSION:PTD-mIFN-? was prepared successfully.