Gene Cloning,Differential Tissue Expression And Antiviral Activities Of Type Ⅰ Interferons Of Bama And Banna Minipigs

Interferons(IFNs)are a group of cytokines with antiviral activities and immune regulatory function.According to their chromosome localization,gene structures,sequence similarities and recognition receptors,mammalian IFNs are classified into three types.As the single member of type Ⅱ IFN,IFN-γ plays a vital role in acquired immunity.In contrast,typeⅠ and Ⅱ IFNs are characterized by multiple genes and play essential roles in innate immunity.With more than 60 functional genes,porcine IFN(PoIFN)complex represents the ideal model for studying IFN evolution resulted from virus pressure during domestication.Among the three types of PoIFNs,type Ⅰ PoIFNs consist of at least 39 functional genes which can be classified into 17 PoIFN-α subtypes,11 PoIFN-δ subtypes and 7 PoIFN-ω subtypes,as well as a single subtype of PoIFN-β,PoIFN-ε,PoIFN-κ and PoIFN-αω.Compared to domestic pigs,minipigs are less domesticated and experienced different virus pressures.Bama and Banna minipigs are the two unique minipig breeds in the world.The two minipig breeds have been developed as the laboratory inbred lines,and used extensively in xenotransplantation,drug toxicity evaluation,virus infection and vaccine evaluation.Innate immunity plays a vital role in disease resistance and IFNs are the major players of innate immunity.However,the PoIFN complex of minipigs remains to be studied.In this study,the PoIFN-αω,PoIFN-β and 17 subtypes of PoIFN-α genes of the two Chinese minipig breeds were cloned,and their sequences,tissue differential expression profiles and antiviral activities were studied,aiming at providing the basic foundation for rationale use of the two minipig breeds.Gene cloning,tissue differential expression and antiviral activities of interferon-αω of Bama minipigsIFN-αω is an emergent subtype of type Ⅰ IFNs which has been primarily characterized in cattle,horses and domestic pigs,but not in minipigs.In this study,the cellular RNA was extracted from different tissues of Bama minipigs.The specific primer pair was designed according to PoIFN-αω gene of domestic pigs,and used to amplify the PoIFN-αω cDNA of Bama minipigs by RT-PCR.After cloning into a plasmid vector,the amplicons were sequenced in two directions for sequence analysis.The PoIFN-αω expression levels in different tissues were detected by real-time quantitative RT-PCR.The coding sequence was cloned into a prokaryotic expression vector and transformed into E.coli.The expression of the His-tagged protein was induced with IPTG and purified by nickel affinity chromatography.Both pre-infection and post-infection antiviral activities of the purified protein against three pig viruses were detected by cytopathic effect(CPE)inhibition assay.The result showed that an expected 558-bp cDNA was amplified from the skin,liver,spleen or lung tissue from Bama minipigs.After transformation into E.coli,a total of 24 clones were sequenced,all of which were identical to the PoIFN-αω cDNA of domestic pigs.The cDNA encoded a polypeptide of 185 amino acids,the first 23 amino acids of which was predicted be the signal peptide.The mature polypeptide contained a putative N-glycosylation site,4 conserved cysteine residues and multiple binding sites of IFNA receptor.Among ten different tissues detected,the expression levels of PoIFN-αω gene in the spleen,kidney,small intestine and lymph node were higher than that in skin,heart,uterus,liver lung and brain.Both expression levels and tissue expression profile of the PoIFN-αω gene of were significantly different from that of domestic pigs.After transformation into E.coli and IPTG induction,an expected 21-kDa protein was expressed as a soluble protein.The recombinant PoIFN-αω(rPoIFN-αω)was purified to a single band and recognizable by the mAb against its C-terminal His-tag.The purified rPoIFN-αω showed dose-dependent pre-infection and post-infection antiviral activities against pseudorabies virus(PRV),vesicular stomatitis virus(VSV)and porcine reproductive and respiratory syndrome virus(PRRSV),but not against porcine circovirus type 2(PCV2),which may be due to the presence of IFN responsive element in PCV2 genome.These experiments demonstrated significant difference in tissue expression profile and antiviral activity of PoIFN-αω between Bama minipigs and domestic pigs,which may be caused by environmental factors and/or immune status.Gene cloning,tissue expression profiles and antiviral activities of interferon-β from two Chinese minipig breedsAlthough the PoIFN-β cDNA sequences of domestic pigs,Chongjiang Xiang pigs,African minipigs and three species of African wild boars are available in GenBank,their tissue expression profiles and antiviral activities remain to be studied.In this study,both genomic and cellular RNA were extracted from different tissues of Bama and Banna minipigs.The gene-specific primer pairs were designed according to PoIFN-β gene of domestic pigs,and used to amplify the PoIFN-β cDNA and its promoter sequence by high fidelity PCR.After cloning into a plasmid vector,the amplicons were sequenced in two directions,and the two consensus sequences were aligned against PoIFN-β sequences of different animal species.The expression levels of PoIFN-β genes in different tissues were detected by real-time quantitative RT-PCR.After cloning into a prokaryotic expression vector and transformation into E.coli,the recombinant protein expression was induced with IPTG.The His-tagged protein was purified with nickel affinity chromatography and identified by Western blotting using the His tag-specific monoclonal antibody.The antiviral activities of the rPoIFN-β against three different pig viruses were detected using CPE inhibition assay.The result showed that the two PoIFN-β cDNAs of the two minipig breeds were identical,which encoded polypeptides of 186 amino acids.The first 21 amino acids were predicted to be the signal peptides,and the mature polypeptides contained one putative N-glycosylation site and three conserved cysteine residues.Sequence analysis showed that the two PoIFN-β sequences shared 97.9-100%homology at nuclear sequence level or 98.4100%homology at amino acid level with that of domestic pigs,Chongjiang Xiang pigs,African minipigs and three species of African wild boars.In addition,the four promoter regulatory domains(PRDs)of the two PoIFN-β genes were identical to that of other six pig species,including IRF3 and IRF7 in PRD Ⅰ and PRD Ⅲ,NF-kB in PRD II,and ATF and Jun in PRD Ⅳ.Among the ten different tissues detected,the two minipig breeds showed significant difference in PoIFN-β gene expression,which were also significantly different from that of domestic pigs.An expected 21-kDa protein was expressed in E.coli as a soluble protein,and purified to a single band,which was recognizable by the mAb against its Cterminal His-tag.The purified rPoIFN-β showed dose-dependent pre-infection and postinfection antiviral activities against VSV,PRV and PRRSV,which were significantly different from that of domestic pig PoIFN-β.These experiments demonstrated the high conservation of PoIFN-β within the Suidae family with different tissue expression profiles and antiviral activities,which may be caused by environmental factors and/or immune status.Gene cloning,differential expression and antiviral activities of interferon-αsubtypes of Bama minipigsPoIFN-α has 17 subtypes and their gene sequences,tissue expression profiles and antiviral activities have been primarily characterized in domestic pigs,but not in minipigs.In this study,both genomic DNA and cellular RNA were extracted from different tissues of Bama minipigs.The gene-common and gene-specific primers were designed according to 17 subtypes of PoIFN-α genes of domestic pigs and used to amplify the PoIFN-α cDNAs and their promoter sequences of Bama minipigs.After cloning into a plasmid vector,the amplicons were sequenced in two directions and their consensus sequences were aligned against that of domestic pigs.The expression levels of different PoIFN-α subtypes in different tissues were detected by real-time RT-PCR.After cloning into a eukaryotic expression vector,the recombinant vectors were transfected into HEK-293 cells.The Histagged proteins were purified by nickel affinity chromatography and identified by Western blotting.The antiviral activities against three different pig viruses were tested using CPE inhibition assay.By using PoIFN-α subtype-common primers,two cDNAs of 546-bp and 570-bp were amplified.After transformation into E.coli,a total of 121 clones were sequenced and seven consensus sequences were obtained,with 98-100%sequence homology with PoIFN-α1,PoIFN-α2,PoIFN-α8,PoIFN-α10,PoIFN-α12,PoIFN-α13 and PoIFN-α16 of domestic pigs.By using PoIFN-α7/11 gene-specific primer pair,a 546-bp cDNA was amplified and two consensus sequences were obtained,which shared 99-100%homology with the PoIFN-α7/11 of domestic pigs.By using PoIFN-α14 gene-specific primer pair,a 570-bp cDNA was amplified and one consensus sequence was obtained,with 99-100%sequence homology with the PoIFN-α14 of domestic pigs.By using the subtype-common primers,the remaining 7 subtypes of PoIFN-α coding sequences were amplified from genomic DNA with 98-100 sequence homology with PoIFN-α3,PoIFN-α4,PoIFN-α5,PoIFN-α6,PoIFN-α9,PoIFN-α15 and PoIFN-α17 of domestic pigs.Sequence alignment showed that 17 PoIFN-α subtypes of Bama minipigs shared 99%-100%homology at nucleotide sequence level or 98%-100%homology at amino acid sequence level.By using the subtype-common or gene-specific primers of PoIFN-α promoters,two amplicons of 98bp and 116bp were amplified and 27 clones were sequenced.The four virus responsive elements in the nine consensus sequences were identical to that of domestic pigs,including IRF3,IRF7 and "TATA" box.Among ten different tissues tested,significant expression of multiple-subtype PoIFN-α was detected in lymph nodes and spleen,while no or low expression of fewer subtypes was detected in heart,lung,brain and small intestine.After transfection into HEK-293 cells,17 subtypes of rPoIFN-α peptides were prepared with significant difference in antiviral activities against VSV,PRV and PRRSV.Both tissue expression profiles and antiviral activities of 17 PoIFN-α subtypes of Bama minipigs differed significantly with that of domestic pigs,which may be caused by environmental factors,immune status and/or detection method.Gene cloning,and differential expression of interferon-α subtypes of Banna minipigsIn this study,the cellular RNA was extracted from different tissues of Banna minipigs.By using PoIFN-α subtype-common primer pairs,two amplicons of 546bp and 570bp were amplified from the pooled cDNA of spleen,liver,lung and small intestine.After cloning into a plasmid vector,171 clones were sequenced in two directions and 17 consensus sequences were obtained,which were shared 98-99.9%homology at nucleotide level or 95-100%homology at amino acid level with 17 PoIFN-α subtypes of domestic pigs.Like that in domestic pigs and Bama minipigs,the intact ORFs of PoIFN-α subtypes in Banna minipigs encoded 189aa polypeptides and those with C-terminal 24-bp deletion encoded 181aa polypeptides.The first 23 amino acids of all PoIFN-α subtypes were predicted to be signal peptides.After cleavage off the signal peptides,the first residues of the mature peptides were always cysteine,with other three conserved cysteine residues at positions 29,99 and 139,as well as a potential N-glycosylation site at position 78.The 17 PoIFN-α subtypes of Banna minipigs shared 96.3-99.8%homology at nucleotide level or 92.3-99.5%homology at amino acid level.Quantitative RT-PCR analysis showed significant difference in tissue expression profiles of PoIFN-α subtypes between Bama and Banna minipigs.For the two minipig breeds,both spleen and lymph nodes were the multi-subtype high expression tissues,while heart and brain were the less-subtype low expression tissues.In contrast,multiple types of PoIFN-α were highly expressed in the skin,lung and small intestine of Banna minipigs,while only some subtypes were expressed at low levels in the three tissues from Bama minipigs.Since the high conservation of PoIFN-α promoters,the detailed reason(s)for different tissue expression profiles of PoIFN-α subtypes between the two minipig breeds remain to be defined.