Amino acid sequence of anti-okadaic acid single-chain antibody and expression vector thereof

[0028] Example 1 Preparation of OA-scFv gene

[0029] 1.1 Total RNA extraction and cDNA synthesis.

[0030] The preparation method of anti-OA monoclonal antibody hybridoma cells is the same as the reference Wang R , Zengl L , Yang H, et al. Detection of okadaic acid (OA) using ELISA and colloidal goldimmunoassay based on monoclonal antibody.[J]. Journal of Hazardous Materials, 2017, 339(oct.5):154-160. The total RNA of anti-OA monoclonal antibody hybridoma cells was extracted using ER501-01 kit (Beijing Quanshijin Biotechnology Co., Ltd.). Using the extracted total RNA as a template, cDNA was synthesized using the K1691 cDNA Synthesis Kit (Thermo Fisher Scientific (China) Co., Ltd.). All operations were performed according to the kit instructions.

[0031] 1.2 Design of primers

[0032] The designed primers are shown in Table 2 and were synthesized by Jinweizhi Company.

[0033] Table 2 Primer sequence list

[0034]

[0035] Note: M=A / C, R=A / G, S=C / G, W=A / T

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[0056] Example 2 pET-MBP-His-OA-scFv expression vector construction and expression identification

[0057] 2.1 Construction of pET-MBP-His-OA-scFv expression vector

[0058] The OA-ScFv gene was used EcoR I and Hind Ⅲ After the double digestion, use agarose gel electrophoresis, and use the gel recovery kit (purchased from ThermoFisher Scientific) to recover the digested products. The pET28a-MBP-T7-6×His vector (purchased from Wuhan Biotechnology Co., Ltd.) containing soluble tag maltose binding protein was also passed through EcoR I and Hind Ⅲ After double enzyme digestion, use agarose gel electrophoresis to recover large fragments. The OA-ScFv gene digestion product fragment was ligated with the pET28a-MBP-T7-6×His vector that had undergone the same double digestion at 16°C for 2 hours (or overnight at 4°C) to construct the recombinant plasmid pET-MBP-His -OA-scFv.

[0059] 2.2 Transformation of recombinant plasmids and expression and purification of recombinant prot