High Level Expression Of Genetically Engineered Tetravalent Antibodies Against Parathion In E.coli And Its Identification

1 To construct expression vector of tetravalent antubodyGene of tetravalent antibody was obtained by PCR after design of primers based on known gene sequence and optimization of reaction conditions with orthogonal test. Preliminary identification was carried on through restriction enzyme and PCR experiment. Positive clones was sequenced by and linked with pTO-T7. Then, expression vector of tetravalent antubody against parathion had been constructed succefully.2 To induce and analyse tetravalent antubodyAfter inducible expression with IPTG, Molecular weight and biospecific characters of recovery product was analysised by SDS-PAGE and identified by Western Blot respectively. Though inclusion body still existed, the analysis results show yield of soluble antibody was enhanced successfully. Ultimate capacity of target protein was 0.29mg/ml ascertained with BCA Assay Kit.3 To identify bioactivity of tetravalent antubodyIn comparison with ScFv, monoclonal antibody and previous tetravalent antibody, result of immunoassay showed specific binding of parathion with affinity constant 3.84×108L/mol and IC50 0.50μg/ml. The detection sensitivity of ELISA was improved for better affinity of tetravalent antibody.4 Presently, there is no clear solution to thoroughly avoid inclusion body for many factors leading to its formation. A combination of codon bias, gene regulatory sequences, inducing congditions and other resons was considered and optimized in this study. It is useful exploration to realize soluble expression of engineering antibody.