Gene construction and expression of human antiª²HBsAg singleª²chain Fvª²Tat fusion protein in E. coli

    MENG Yanª²Ling, WEN Weiª²Hong, XUE Qian, JIA Linª²Tao, BAO Wei, LIU Jiaª² Yun, REN Junª²Lin, XUE Caiª²Fang, LI Yingª²Hui, WANG Chengª²Ji, YANG Anª²Gang

    1Department of Immunology, 2Department of Biochemistry and Molecular Biology, 3Department of Etiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct a fusion gene of human antiª²HBsAg ScFvª²Tat and to analyze the binding activity and internalization of ScFvª²Tat fusion protein after its expression in E. coli. METHODS: Two oligonucleotide primers were designed and used to amplify the ScFv gene. The construction was cloned into expression vector pTATª²HA containing the protein transduction domain Tat. The expressed protein was detected by SDSª²PAGE and Western blot and purified by affinity chromatography. The binding specificity of the ScFvª²Tat fusion protein was confirmed by indirect ELISA and specific internalization of the ScFvª²Tat fusion protein was confirmed by immunocytochemistry staining after the purified protein was incubated with HBsAg positive or negative hepatocellular carcinoma cells. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly cloned into expression vector. SDSª²PAGE and Western blot analysis showed that ScFvª²Tat fusion protein was successfully expressed in E. coli BL21. Indirect ELISA and competition inhibition ELISA confirmed that the expression products had antigen specific binding activity. ScFvª²Tat fusion protein was specifically internalized into HBsAg positive hepatocellular carcinoma cells. CONCLUSION: The antiª²HBsAg ScFvª²Tat fusion protein expressed in E. coli can specifically internalize into HBsAg positive hepatocellular carcinoma cells. ......