Study On Anti-canine Parvovirus Infection Of Recombinant Canine Soluble Transferin Receptor

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). The disease is mainly manifested in acute hemorrhagic gastroenteritis and acute myocarditis with high morbidity and mortality, which is harmful to dogs, in particular to puppies, therefore it is an important infectious disease .It has been proved that transferrin receptor (TfR) on the host cell membrane are parvovirus receptors of dogs, cats and other animals. The TfR can bind capsid protein VP2 of parvoviruses and mediate the virus enternalization into the cells.We attempted to use recombinant soluble TfR protein (sTfR) to inhibit or block the CPV infection to host cells. In order to prepare a large amount of recombinant sTfR, the sTfR gene codons were optimized to increase sTfR expression level in eukaryotic cells. First, the sTfR gene was amplified from canine liver tissues by RT-PCR. According to amino acid sequence, sTfR codons were optimized based on human main codons and synthesized by the company. The eukaryotic expression vectors either for wild-type sTfR gene and codon-optimized sTfR genes were construced by using pcDNA3. 1-CD5 vector. The secretory expression vector of sTfR gene fused with His-tag at C-terminal and fused with human CD5 signal peptide in N-terminal was constructed using pcDNA3.1A-CD5 plasmid. The vectors were t ransfected into human embryonic kidney cells HEK293T cells mediated by calcium phosphate. The expressed sTfR was purified by Ni-agarose affinity chromatography from culture media and was identified by Western-blot. We analyzed sTfR binding activities to CPV-2a and its VP2 protein by using ELISA method and evaluated anti-CPV infection of sTfR in feline kidney F81 cells.The result s showed that the amplified sTfR gene sequence was 100 % similarity to the published in GenBank. sTfR gene with CD5 signal peptide could be secretively expressed in HEK293FT cells. The codon optimization could significantly enhance sTfR gene expression compared with wild-type sTfR gene. The expression levels of the codon-optimized sTfR gene was 2.6μg/106 cells,more than 1-fold increased compared with that of wild-type construct (1.2μg/106 cells). The recombinant sTfR possessed the ability of specific binding to CPV and CPV-encoded VP2 proteins. The sTfR could significantly inhibit the infection of CPV in F81 host cells. Those results indicate that the sTfR protein possesses antiviral activity to canine parvovirus, which laids the foundation for further investigation of sTfR anti-CPV activity with dogs in vivo and exploration of new way to develop anti-viral agents.