title: Generation and characterization of a fusion protein of single-chain fragment variable antibody against hemagglutinin antigen of avian influenza virus and truncated protamine.
abstract: The hemagglutinin antigen (HA) of avian influenza virus (AIV) is an immunogen abundant on the surfaces of infected cells, and can be used as a target for specific antibodies to clear viral infection. Protamine has been demonstrated to deliver DNA into cells effectively. Accordingly, a fusion protein of anti-HA single-chain fragment variable (scFv) and truncated protamine (tP) may be used as a vehicle for delivering the anti-AIV siRNA into the AIV-infected cells for gene therapy. To test this hypothesis, we constructed a novel recombinant plasmid, pET28-scFv-tP, by connecting the genes for anti-H5N1 AIV HA-specific scFv with synthesized oligonucleotides encoding the 22 amino acids of human tP and a linker. Furthermore, the recombinant scFV-tP was expressed and purified, with a yield of 7-8mg of scFv-tP and a purity of >92% from 1L of bacterial culture. Characterization of its bioactivity revealed that scFv-tP recognized HA, similar to its scFv control, in a dose-dependent manner and that the scFv-tP, but not its scFv control, bound to DNA and delivered plasmid and oligonucleotide DNA into the AIV-infected MDCK cells effectively. More importantly, transfection with the mixture of the scFv-tP and plasmid for the NP-specific siRNA significantly inhibited the replication of AIV in MDCK cells, as compared with that transfection with the scFv-plasmid mixture, even with the plasmid in liposome. Our data demonstrated that the recombinant scFv-tP retained the functions of both scFv and tP, and might be potentially used for delivering genetic materials for targeting therapy of AIV infection in vivo.
authors.affiliation: Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China. zhangtao1012450@gmail.com
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Gao
authors.forename: Song-Tao
authors.initials: ST
authors.lastname: Yang
authors.forename: Tie-Cheng
authors.initials: TC
authors.lastname: Wang
authors.forename: Ren-Zhou
authors.initials: RZ
authors.lastname: Zhang
authors.forename: Chuan
authors.initials: C
authors.lastname: Qin
authors.forename: Xian-Zhu
authors.initials: XZ
authors.lastname: Xia
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D004285:Dogs; D015316:Genetic Therapy; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology* / Q000502:physiology; D009976:Orthomyxoviridae Infections / Q000276:immunology; D010957:Plasmids; D011479:Protamines / Q000276:immunology*; D034741:RNA, Small Interfering / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis*; D019444:Vaccines, DNA / Q000276:immunology; D014779:Virus Replication
chemical_list: D000956:Antigens, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011479:Protamines; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D019444:Vaccines, DNA