Cloning And Expression Of Siniperca Chuatsi IFN-?3 Gene And Preparation Of Polyclonal Antibodies

Objective: Interferons are a multifunctional protein,it is a cytokine produced by lymphocytes and monocytes when the body and cells are stimulated by viruses or other inducers,can induce vertebrates to enter the antiviral state.In addition to antiviral activity,it participates in other important physiological processes,such as regulating cell growth,differentiation,apoptosis and immune response.Fish is a lower vertebrate,and non-specific immunity plays an important role.As an efficient non-specific immune factor,interferon system plays an important role in the anti-infective immunity of fish.Siniperca chuatsi is an important freshwater aquaculture species in China,with the increase of aquaculture density,the disease is more serious,outbreak of infectious spleen and kidney disease caused by ISKNV,it not only brings huge economic losses,but also restricts the development of the Siniperca chuatsi culture industry.The aim of this study was to clone the IFN-?3 gene,the expression pattern of IFN-?3 was analyzed,the prokaryotic expression vector of IFN-?3 was constructed,the recombinant protein was induced to produce specific polyclonal antibody,to reveal the expression of IFN-?3 gene and IFN-?3protein in Siniperca chuatsi after Poly I: C stimulation,it lays the foundation for the functional study of IFN-?3,the research on the prevention and treatment of fish viral diseases has important theoretical and practical significance.Methods: The rapid amplification of c DNA ends(RACE)procedure was used to obtain the full length c DNA of Siniperca chuatsi IFN-?3,the relative expression of IFN-?3in Health and Poly I: C Stimulation of Siniperca chuatsi various tissues was detected by q PCR,the p ET32a(+)prokaryotic expression system was used to express IFN-?3recombinant protein,purified IFN-?3 recombinant protein as the immunogen,and an anti-rgc IFN-?3 polyclonal antibody was prepare by immunizing mice with the purified rgc IFN-?3 alternately via intraperitoneal and subcutaneous injections.The titer of antibody was detected by double immunodiffusion assay and enzyme-linked immunosorbent assay(ELISA),the specificity of antibody was detected by Western blot,finally,western blot was used to analyze the expression characteristics of natural IFN-?3 protein in various immune-related tissues of the Siniperca chuatsi after the stimulation with Poly I:C.Results: The full-length c DNA of Siniperca chuatsi IFN-?3 is 863 bp long,containinga 537-bp open reading frame that encoded a peptide of 178 amino acids,after the signal peptide was removed,the predicted molecular weight was 18 k Da,the amino acid sequence contained a signature that is proposed to be characteristic of the IFN-?3 protein family.Phylogenetic tree analysis showed that,IFN-?3s from Siniperca chuatsi and other members of the Perch family clustered into a single group.Healthy Siniperca chuatsi thymus,head kidney,gill,spleen,liver,intestine,body kidney and blood,all 8 tissues of IFN-?3 m RNA have different levels of expression,the transcription level of blood,head,kidney,spleen and thymus is higher than that of other tissues,after Poly I: C stimulated fish,the level of IFN-?3 transcription was significantly up-regulated in thymus,head kidney,spleen,liver,body kidney and blood;Among them,liver up-regulated the peak of IFN-?3 after Poly I: C stimulation for 3 h,thymus was the highest expression at 6h after Poly I: C stimulation,the head kidney and spleen reached the highest peak at 12 h and 48 h respectively.The expressed strain was induced by IPTG and the expressed product was detected by SDS-PAGE.The result showed that the size of the recombinant protein was 36 k Da,which was consistent with the predicted size.The double immunodiffusion assay results showed that the titers of the polyclonal antibody is 1:32,the enzyme-linked immunosorbent assay results showed that the Siniperca chuatsi IFN-?3 polyclonal antibody titer up to 1: 64 000;Western blot results show,the prepared polyclonal antibody can specifically recognize the prokaryotic expressed IFN-?3 recombinant protein and the natural IFN-?3 protein in Siniperca chuatsi.Conclusion: We have cloned and identified the Siniperca chuatsi IFN-?3 gene,the expression patterns of IFN-?3 in Health and Poly I: C Stimulation of Siniperca chuatsi various tissues were analyzed.Induced expression of recombinant protein,anti-rgc IFN-?3polyclonal antibody was prepared and verified,the study confirmed that,after stimulation with Poly I: C,the Siniperca chuatsi expressed the IFN-?3 protein.This study provides research ideas for the prokaryotic expression of proteins and the preparation and validation of specific polyclonal antibodies,specific antibodies were also provided for the study of the function of the IFN-?3 gene,at the same time,it lays a foundation for the development and utilization of fish interferon in the prevention and control of fish diseases.