Generation and characterization of a fusion protein of single-chain fragment variable antibody against hemagglutinin antigen of avian influenza virus and truncated protamine.
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.
Zhang
Tao
T
Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China. zhangtao1012450@gmail.com
Wang
Cheng-Yu
CY
Zhang
Wei
W
Gao
Yu-Wei
YW
Yang
Song-Tao
ST
Wang
Tie-Cheng
TC
Zhang
Ren-Zhou
RZ
Qin
Chuan
C
Xia
Xian-Zhu
XZ
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
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