Cloning,bioinformatics Analysis And Expression Optimization Of Panthera Pardus Fontanierii Interferon-? Family

Type ? interferons are key anti-viral cytokines that have evolved during the formation of vertebrate species to respond to various viral threats.Interferon-?(IFN-?)is a glycoprotein produced by leukocytes against viruses,bacteria or other foreign invaders,which has various biological functions such as anti-virus reproduction,anti-tumor growth and immune regulation.At present,the research of animal IFN has developed rapidly.Commercially available recombinant IFN products such as chicken,dog and pig have been applied in the market.However,there are few reports on IFN in the Panthera pardus fontanierii(North China leopard)at home and abroad.The Panthera pardus fontanierii is a unique subspecies of leopard in China,also known as Chinese Leopard.Due to habitat destruction,human killings and the spread of viral diseases,the number of Panthera pardus fontanierii has shown a sharp decline,and it has been included in the IUCN Red List of Endangered Species.The broad-spectrum antiviral effect of IFN is likely to become a new approach and method for the prevention and treatment of Panthera pardus fontanierii virus disease,and it also meets the current natural ecological,pollution-free,non-residue,and environmental protection standards.Based on the Panthera pardus IFN-? gene sequence published by GenBank,we designed and synthesized a pair of specific primers.Using the Panthera pardus fontanierii nucleic acid DNA as a template,11 Panthera pardus fontanierii IFN-? subtype genes(PfIFN-?/?1/?2/?3/4/?5/?6/?7/?8/?9/?10)were amplified for the first time.Sequence analysis showed that PfIFN-?/?1/?2/?3/4/?5/?6/?7/?8 open reading frame lengths are 570bp in length and encode 188 amino acids.The open reading frames of PfTFN-?9 and PfIFN-?10 are 567bp,which encode 187 amino acids.The signal peptide is a hydrophobic transmembrane domain structure composed of 23 amino acids,and the mature peptide region is mainly a hydrophilic protein without transmembrane domain.They all contain 6 conserved cysteine residues that located at 1AA,29AA,69AA,87AA,100AA and 138AA in the mature peptide sequence.The nucleotide homologies of 11 PfIFN-?s were 97.4%-99.8%,and the amino acid homologies of 11 PfIFN-?s were 93.7%-98.9%.The protein structures of PfIFN-?s were mainly composed of 5 ?-helix(accounting for more than 60%).Homology and phylogenetic tree analysis showed that the IFN-? subtype genes of Panthera pardus fontanierii have high homology with mammalian,especially carnivora IFN-? gene,and the genetic distance are relatively close.The mature peptide sequences of the PfIFN-? subtype gene were connected to the prokaryotic expression vector pET32a(+)to construct a recombinant expression plasmid pET32a-PfIFN-?s,which were transformed into E.coli TSsetta(DE3).SDS-PAGE electrophoresis analysis showed that the expression products mainly existed in the form of inclusion bodies,and the recombinant protein obtained by purification and renaturation was about 37KDa.Optimizing its expression conditions,the recombinant protein was induced to express at a concentration of 0.8 mM IPTG for 6 hours to obtain the optimal expression level.Using the purified PfIFN-?recombinant protein as immunogen,we prepared rabbit anti-PfIFN-? polyclonal antibody.The antibody titer of enzyme linked immunosorbent assay was 1:204800.Westernblot showed that the rabbit anti-PfIFN-? polyclonal antibody could specifically recognize PfIFN-?s recombinant protein.In summary,this study successfully cloned 11 PfIFN-?s genes,constructed their recombinant prokaryotic expression plasmids and realized the expression,purification and renaturation of recombinant proteins,providing material support for the subsequent physical and chemical properties and biological activities of PfIFN-?s.