Dissertation > Environmental science, safety science > The basic theory for the Environment and Science > Environmental Biology > Ecosystems and pollution ecology > Pollution Ecology

Cloning, Identification and Eukaryotic Expression of Variable Region of Monoclonal Antibodies Against Chelated Mercury, Copper and Zinc and Three Dimentional Modeling of Recombinant Antibody

Author LiPeng
Tutor LiuFengQuan
School Nanjing Agricultural College
Course Plant Pathology
Keywords monoclonal antibody against heavy metal sequence identification eukaryotic expression protein-protein interaction three dimentional modeling
CLC X171.5
Type Master's thesis
Year 2011
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Heavy metals can lead to side effects even if they are present in extremely minute quantities because of their nonbiodegradable nature and ability to persist for long periods. Enzyme-linked immunosorbent assay (ELISA) developed for monitoring of trace heavy metals in food and environment because of its rapid, sensitive and simple characteristics is favored. Many antibodies directed toward metal-chelate complexes are being developed, however, except for some liqud sample such as water, they can not be applied to the real environmental and food detection, mainly in the preparation of high cost, cross-reactivity and so on. The solution to this problem depends on the depth study of molecular mechanism of antibody binding antigen by genetic engineering.Hybridoma cellines which secrete monoclonal antibody against heavy metals chelators were selected in our lab.3G4G5 is the monoclonal antibody against with mercury (Ⅱ)-ITCBE,9D11 is the monoclonal antibody against with copper(Ⅱ)-isothiocyanatobenzyl-DTPA (SCN-DTPA) chelator (Cu(Ⅱ)-DTPA),2E8E3 is the monoclonal antibody against zinc(Ⅱ)-SCN-DTPA chelator(Zn(Ⅱ)-DTPA). Primers were designed and synthesized to contain the signal peptide, whole variable region segments and partial constant region including Cys. VH and VL of three monoclonal antibodies were cloned by RT-PCR method. After sequencing, the DNA sequences were analyzed by BLAST, SignalP analysis and IMGT-V/QUEST analysis. The results showed that these were homologous with murine antibodyμchain orκchain. The leading sequences were with character translated to be the signal peptide, the specific-site-cysteines in antibody were also in the clonal DNA sequence. Three eukaryotic recombinant expression vectors, pBudCE4.1-3G4G5, pBudCE4.1-9D11 and pBudCE4.1-2E8E3 and two reporter vectors were constructed. The optimized transfection system was detemined by transiently transfecting eukaryotic cell lines with the reporter vectors under different conditions. Recombinant antiobodies were expressed on the optimized transfection system and were purified simply, and the active of antibodies was determined by ELISA. In order to study whether VH and VL of recombinant antibody could combine together by an interchain disulfled bond, a BiFc (bimolecular fluorescence complementation) vector named pBudCE4.1-VHCEYFP-VLNEYFP was constructed and subsequently was used to transfect 293T cell lines. Three dimentional models of monoclonal antibodies of 3G4G5,9D11 and 2E8E3 were constructed by computer on Swiss-Model Server. The VH and VL of antibodies were homology modeled respectively and then they were merged as the whole three dimentional models. The whole models were refined by the motheds of molecular mechanics and dynamics. The Procheck and Verify-3D were used to evaluate the models. The experiment shows that the structure of models was reasonable reliable. Key amino acids residues were predicted by the comparison of antibody models and the crystallographic structure of CHA255, a previous reported antibody directed toward In-EDTA.All data showed that the correct variable region sequences of both antibodies 3G4G5, 9D11 and 2E8E3 were cloned, the correct expression vectors were constructed and the active recombinant antibodies were got by transient transfection of 293T cell lines, and three dimentional models of those antibodies were constructed successfully. This study provided a method for improving antibody affinity and a good platform for the molecular mechanism of antigen-antibody binding theory.

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