Authors: Xixia Liu Hong Wang Yan Liang Jinyi Yang Hongbin Zhang Hongtao Lei Yudong Shen Yuanming Sun
Publish Date: 2010/01/20
Volume: 45, Issue: 1, Pages: 56-64
Abstract
The production and characterization of an anticlenbuterol singlechain Fv antibody CBLscFv–bacterial alkaline phosphatase AP fusion protein are described The CBLscFv and the phoA gene of Escherichia coli strain K12 chromosomal DNA were cloned by PCR and sequentially inserted into the expression vector pBV220 to express the CBLscFv–AP fusion protein in E coli strain BL21DE3pLysS SDS–PAGE and western blot analyses revealed that the fusion protein showed a molecular weight of 73 kDa and bound with the antibacterial AP monoclonal antibody Determination of enzymatic activity indicated that k cat and K m values of the fusion protein were 11360 s−1 and 2982 μM respectively Competitive direct enzymelinked immunosorbent assay based on the obtained fusion protein indicated that the average concentration required for 50 inhibition of binding IC50 and the limit of detection for CBL were 474 ± 0003 n = 3 and 054 ± 0004 n = 3 μg/l respectively and the linear response range extended from 113 to 6968 μg/l Crossreactivity studies showed that the fusion protein did not crossreact with CBL analogs The present findings indicate that the production of the CBLscFv–AP fusion protein in E coli strain BL21DE3pLysS is feasible and suggest that it could be further used to develop a onestep ELISA for the specific detection of CBL
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