Preparation,Structural And Functional Analysis Of Monoclonal Antibodies Against Type Ⅱ Interferon In Grass Carp(Ctenopharyngodon Idella)

Type Ⅱ interferon,also termed IFN-γ(IFN-γ),is one of the key cytokines involved in the immune response of T helper 1(Th1)lymphocytes.Fish and mammalian type ⅡIFN-γs are homologs,but unlike mammals which contain a single copy of IFN-γ gene,teleost fish have two duplicated copies,namely IFN-γ and IFN-γ related(IFN-γrel)molecule.To date,the expression and functions of fish IFN-γs have been well documented,however fish IFN-γrels have not been extensively investigated.For example,the cells producing IFN-γ and IFN-γrel are unclear,whether they have differences in functions has also been debated.The recombinant IFN-γ and IFN-γrel protein of grass carp(Ctenopharyngodon idella,Ci)were expressed in bacteria.It was found that the IFN-γ was expressed as soluble protein in DE3 cells whilst IFN-γrel as inclusion bodies.The recombinant proteins were purified using affinity chromatography and size exclusion chromatography for generation of monoclonal antibodies and characterization of biological activities.The eukaryotic recombinant Ci IFN-γ/IFN-γrel proteins were expressed in eukaryotic system CHO-S and HEK293 F cells,respectively.After HKLs were stimulated,the regulatory effects of IFN-γ and IFN-γrel on immune genes were detected by q PCR.By expressing Ci IFN-γrel selenoprotein,the crystal structure of Ci IFN-γrel was analyzed.The binding relationship between Ci IFN-γrel and the receptor was verified by Co-IP experiment.The specific research results are as follows:(1)In this study,the recombinant IFN-γ and IFN-γrel proteins were obtained and used for generation of monoclonal antibodies in mice.Four antibodies with high specificity and affinity were obtained after screening by Western Blotting.It was revealed that the monoclonal Ci IFN-γ antibodies did not cross-react with the Ci IFN-γrel protein and vice versa.Two of the antibodies were labeled with FITC fluorescein and could be used for immunofluorescent analysis and flow cytometry.Further,grass carp IFN-γand IFN-γrel monoclonal antibodies could not recognize their respective homologs from zebrafish.(2)To investigate whether IFN-γ and IFN-γrel differentially modulate immune gene expression,the primary HKLs were stimulated with different doses of recombinant IFN-γ and IFN-γrel proteins purified from the eukaryotic cells for 6 h and 12 h.It was found that Il-1β and Cxcl8-l1 were upregulated by both IFN-γ and IFN-γrel.In contrast,Il-6 did not respond to IFN-γ and IFN-γrel stimulation.Cxcl11.b,Mhc II and T-bet genes were markedly induced by IFN-γ but curiously not by IFN-γrel.In additition,both IFN-γ and IFN-γrel failed to alter the expression levels of Stat1 a.(3)This study resolves the crystal structure of Ci IFN-γrel,a homodimer with identical peptide chains that are connected by a non-crystallographic two fold axis.Each peptide chain is composed of six α helices,denoted A to F,connected by loops of different lengths(designated by the connecting helices).The dimer structure mainly rely on disulfide bonds and intermolecular hydrophobic forces to maintain the stability of the dimer structure.Compared with other helices,the C helix provides the largest embedding area(BSA).(4)Co-IP results showed that IFN-γrel was co-immunoprecipited with CRFB13 and CRFB17 but had relatively stronger binding affinity with CRFB17 than CRFB13.However,no binding of IFN-γrel with CRFB6 was detecte.Moreover,we observed that CRFB6 could directly bind to CRFB13 and CRFB17.Compared to the wild type IFN-γrel,mutations of Gln23 A,Thr31A,Glu76 A and Asp128 A resulted in weakened binding with CRFB13 while conversely,mutations of Gln45 A and Lys93 A had opposite effects.In contrast,mutant Arg29 A had comparable binding affinity with the wild type IFN-γrel.As for the interaction of IFN-γrel with CRFB17,Asp128 A significantly decreased the binding affinity with CRFB17 while other residues had no effect.(5)The phosphorylation of STAT1 a was increased in the cells stimulated with Ci IFN-γrel in a dose dependent manner.We observed that the STAT1 a phosphorylation levels were decreased in the cells treated with Arg29 A,Thr31A,Gln45 A,Lys93A or Asp128 A mutants compared to wildtype Ci IFN-γrel,with marked inhibition detected for Gln45 A.In contrast,Gln23 A and Glu76 A did not alter the STAT1 a phosphorylation level.QPCR results showed that Lys93A and Asp128A drastically consistently diminished the ability of Ci IFN-γrel in inducing the expression of Il-1βand Cxcl8-l1.In conclusion,high-purity recombinant IFN-γ and IFN-γrel proteins were obtained in this study and used for generation of monoclonal antibodies in mice.Four antibodies with high specificity and affinity were obtained by Western Blotting screening,which can be used for immunofluorescence and flow cytometry analysis.The functional differences between IFN-γ and IFN-γrel were verified,and the crystal structure of Ci IFN-γrel was analyzed,laying a foundation for the in-depth study of the production,biological function and structural biology of type II interferon in grass carp.