Construction and expression of human antiª²HER2 ScFv/Ck/tP fusion protein in E.coli and identification of its actiª²vity

    WANG Kai, WEN Weiª²Hong, WANG Tao, ZHANG Rui, QIN Weiª²Wei, LEI Xiaoª²Ying, YANG Anª²Gang

    1Department of Biochemistry and Molecular Biology,2Department of Immunology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct a fusion protein gene of human antiª²HER2 ScFv/light chain constant region/truncated protamine (ScFv/Ck/tP) and analyze the binding activity of the expressed protein. METHODS: Two pairs of oligonucleotide primers were designed and used to amplify the ScFv and the Ck gene. After they were linked as ScFv/Ck, the synthesized tP coding sequence was added in the 3¡ä terminus of it, then the fusion protein gene ScFv/Ck/tP was cloned into expression vector pET32a and expressed in E. coli BL21 (DE3). Expressed protein was detected by SDSª²PAGE and Western Blot and purified by Niª²NTA chelating agarose. The antigenª²binding activity of the ScFv/Ck/tP fusion protein was confirmed by cellular ELISA, and the DNA binding ability was confirmed by gel shift assay. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that ScFv/Ck/tP was correctly cloned into expression vector. SDSª²PAGE and Western Blot analysis showed that ScFv/Ck/tP fusion protein was successfully expressed in E. coli BL21. Cellular ELISA confirmed that it had specific antigen binding activity; gel shift assay assured that it had DNA binding ability. CONCLUSION: The ScFv/Ck/tP fusion protein expressed in E. coli could specially bind with both HER2 antigen and DNA. ......