Expression Of ChIRF7 And Development Of Monoclonal Antibody Against ChIRF7

IRF7(Interferon regulate factor 7),a member of IRFs family,participates in adaptive and innate immunity regulation and plays a key regulatory role in the expression of type I IFN genes.The pattern recognition receptor could activate the host's different signaling pathways via the interaction with the pathogen-associated molecular pattern after the pathogen invasion.In this case,IRF7 is phosphorylated and activated,moving from the cytoplasm to the nucleus in order to bind to the IFN promoter Meanwhile,IFN molecules is activated and released to activate the JAK/STAT signaling pathway which induces the production of ISGs to defense pathogen invasion.It has been found that the virus could replicate in the mammals by disabling IRF7 which inhibited the hosts,antiviral ability.However,very few papers about how the virus acts on IRF7 to escape the immunity response in poultry have been reported.The aim of this study was to construct prokaryotic and eukaryotic expression plasmids of chIRF7 and generate anti-chIRF7 monoclonal antibody,which could provide bio-materials for the function investigation of chIRF7 in IFN signaling pathway1.Cloning and expression of chIRF7In order to obtain prokaryotic and eukaryotic expression plasmids of chlRF7,total RNA of avian lymphocytes was extracted and reverse transcribed into cDNA,and amplified by PCR with two pairs of primers according to published sequence from GenBank.Then chIRF7 gene was cloned into pCOLD-TF and pcDNA3.1-rIgG respectively.The pCOLD-TF-chIRF7 expression plasmid was transformed into Ecoli BL21(DE3)and induced with 0.25 mmol/L IPTG at low temperature for 20 h.SDS-PAGE and Western-blot analysis showed that fusion protein HIS-chIRF7 was expressed and its molecular weight was about 107 kDa.The eukaryotic expression plasmid was transfected into DF-1 cells and IFA results showed that chIRF7 protein was expressed.2.Development of monoclonal antibody against chIRF7To generate monoclonal antibodies against chIRF7,6-week-old BALB/c mice were injected with fusion protein HIS-chIFR7 expressed in Ecoli,once per 10 days,and cell fusion was performed after the fourth immunization.The pcDNA3.1-chIRF7-rIgG was transfected into DF-1 cells for monoclonal antibody screening.IFA and Western blot analysis both showed that four kinds of hybridoma cells,named as chIRP7-1F12,chIRF7-2B7,chIRP7-5A2,and chIRF7-6G7 respectively,could stably secrete monoclonal antibodies against chIRF7.The ascites titers of four kinds of monoclonal antibodies were all above 1:128000 by IFA.Ig subclass of these monoclonal antibodies showed that chIRF7-1F12 was IgG2b and others were IgG1.The five eukaryotic expression plasmids of partial sequences of chIRF7 were constructed and transfected into DF-1 cells respectively to analyze the epitopes of four kinds of monoclonal antibodies by IFA.The results showed that the epitopes of chIRF7-1F12,chIRF7-2B7,chIRF7-5A2 and chIRF7-6G7 were respectively located at 36-115aa,184-247aa,36-115aa and 142-182aa of the full-length protein of chIRF7.Our research laid a foundation for further study in the regulation mechanism of the expression of chIRF7.