Construction, Expression And Functional Identification Of Hantavirus ScFv/Cκ/truncated Protamine Fusion Proteins

AIM:Hantavirus (HV) infection is one of the most prevalent viral infections of humans. HV infections cause hemorrhagic fever with renal syndrom(eHFRS). There is no effective way can eradicate the virus. In our study, we construct, express and purify a targeted delivery fusion protein, which is composed of a single-chain antibody fragment ScFv that recognizes Hantavirus nucleocapsid or envelope glycoprotein, a constant region of light chain (Cκ) and a truncated protamine, and to analyze the functional activity of the fusion protein.METHODS: Pairs of oligonucleotide primers were designed and used to amplify the ScFv and the ScFv-Cκ. The truncated protamine fragment coding sequence was added in the 3'terminus primer. The fusion protein gene ScFv-Cκ-tP was then cloned into expression vector pET28a, pET32a and expressed in E. coli BL21 (DE3). Expressed protein was detected by SDS-PAGE and Western blot and purified by Ni-NTA chelating agarose. The antigen-binding activity of the ScFv-Cκ-tP fusion protein was confirmed by ELISA, and the DNA binding ability was confirmed by electrophoretic mobility shift assay (EMSA). Internalization ability analyses of the fusion proteins by indirect immunofluorescence staining.RESULTS: HV ScFv-Cκ-tP encoding sequence was correctly cloned into the expression vector. The fusion protein was soluble expressed in E .coli BL21 induced by IPTG. ELISA confirmed that it had specific antigen binding activity; EMSA assured that it had DNA binding activity. Direct immunofluorescence staining confirmed that it can internalization into HV infect cell.CONCLUSION: The ScFv-Cκ-tP fusion protein can specially bind with both HV antigen and DNA. It can internalization into HV infect cell.