Molecular Cloning And Characterization Of IRF1 And IRF2 In Cherry Valley Duck

Interferon(IFN) plays an important role in the anti-viral immune response, and it is also involved in the cell growth, differentiation and immune regulation as an important cytokine.Interferon regulatory factor(IRF) is very important in the transcription of IFN and interferon inducible genes(ISGs).IRF family is an important transcription factor, which can regulate the immune response,especially against virus infections, including induction of IFN and complement activation and natural killer cells, and enhance immune response. It is also participate in hematopoietic response. IRFs owns 11 members(IRF1-IRF11), which detected in mammals and birds out10 members, each them named as IRF1, IRF2, IRF3, IRF4/PIP/LSIRF/ICSAT, IRF5, IRF6,IRF7, IRF8/ICSBP, IRF9/p48/ISGF3γ and IRF10, the IRF11 was found only in fish at present.These members have the same aconserved region in structure: a DNA binding domain(DBD);an IRF association domain in C terminus(IAD, except the IRF1 and IRF2). but different in function. IRF4 and IRF8 can increase telomerase activity through immune cells; IRF7 is a main regulator of IFN; IRF9 can identify some special nucleotide sequence of interferon regulatory factor 3(ISGF3) complexes. The identification of ISGF3 is a key process in the IFN response to viral stimulation; IRF10 can regulate guanylate binding protein expression which similar to IRF1; IRF5 can bind to the IFN and ISGS promoter. IRFs can be divided into four subfamilies by the evolution: IRF1 subfamily(IRF1, IRF2 and IRF11), IRF3subfamily(IRF3 and IRF7), IRF4 subfamily(IRF4, IRF8, IRF9 and IRF10) and IRF5subfamily(IRF5 and IRF6). And divided into three categories in function: activator(IRF1,IRF3 and IRF9), inhibitor(IRF8), double function(IRF2, IRF4, IRF5 and IRF7).IRF1 is a great candidate gene for the study of animal disease, and is also a transcription cytokine which with multiple biological functions. It is involved in the regulation of anti-viral response, cell growth and apoptosis. IRF2 can contact other members of the IRF family and form a complex system of IRF regulation, it effects the antigen presentation, cell cycle, aswell as inhibit the tumor effect; but the function of IRF1 and IRF2 is still unclear in duck.As a very important species in the livestock industry, ducks produce meat(mainly in Asia) 300 million tons per year with a high economic value. Huge economic losses caused by the virus infection. So it is necessary for us to understand the antiviral mechanism to improve the antiviral function in ducks.In this experiment, we selected Cherry Valley Duck as the research object and the research content mainly includes three parts:First part: The cloning and phylogenetic analysis of IRF1 and IRF2 in Cherry Valley Duck.The primers were designed according to the conserved region of IRF1 and IRF2 which published on NCBI. The total RNA was extracted from the normal cherry valley duck in spleen. And the Open Reading Frame(ORF) of IRF1 and IRF2 were cloned through Polymerase Chain Reaction(PCR) amplification method. The length of IRF1 ORF was954 bp, encoding 317 amino acids. ORF of IRF2’s length was 1047 bp, encoding 348 amino acids. The software MEGA5.1 was used to analysis the species of IRF1 and IRF2. Our results showed that the Cherry Valley Duck has the highest homology compared with the swan goose(Credibility of IRF1 and IRF2 were 93% and 78%).Second part: The analysis of IRF1 and IRF2 with other species of amino acid comparative analysis and biological function of Cherry Valley Duck.By online software and review of the related literature, we compared the duck, chicken,mice, human’s IRF1 and IRF2 sequence. The results showed that the four species of amino acid sequences are highly homologous. Referring to other literatures, we also found that IRF1 can be divided into five regions: N-terminal repressor 、 DNA binding domain, nvclear localization, transactivation domain and enhancer domain. IRF2 can also be divided into four areas: DNA binding domain, transactivation domain, IFN associate domain 2 and repression motif.Third part: The distribution of IRF1 and IRF2 in various tissues and expression rules after injected with virus(DTMUV, DPV, NDRV) in Cherry Valley Duck.Real-time PCR(qRT-PCR)analysis results showed that the cherry valley duck IRF1 and IRF2 gene were expressed in all detected tissues, but the expression levels were different.Under normal circumstances, the highest expression of IRF1 was detected in the lung and the highest expression of IRF2 was detected in the heart, liver and lung. In order to study determined the IRF1 and IRF2 are involved in the antiviral immune responses in cherry valley duck. We intramuscular injection DTMUV/DPV/NDRV. Then the expression levels of IRF1 and IRF2 genes in the spleen and brain were detected in 1、3 and 5 days after infection.The results show that the cherry valley duck IRF1 are rapidly upregulated after injection of the virus in 1, 3, 5 days, which DPV stimulated spleen and brain are in 1, 3, 5 days increase ratio gradually increased, on the fifth day reached maximum value(spleen was 7.33 times,the brain to 7.95 times). After stimulation of the NDRV, at the 1, 3, and 5 days showed up-regulation, reaching a peak in the first day(spleen is 234.5 times, the brain is 20.3 times).DTMUV stimulation, up-regulation is not very obvious and under the following 10 times in the spleen; significantly upregulated at 1 day, 3 days, 5 days have reached more than 100 times, of which the first day reached maximum 459.2 times in the brain.After the stimulation of DPV, cherry valley duck IRF2 expression in brain and spleen were up-regulated on the first day and third day. The up-regulation of multiple relatively stable spleen is about 35 times and brain up about seven times; the five days to reach the maximum, spleen increased 151 times, brain raised 15 times. After the stimulation of the NORV, spleen IRF2 reached the maximum up-regulation to 234.5 times, third days and fifth days also known as the upward trend, but the increase in the multiple gradually weakened.The brain reached its maximum up to 8.6 times in third days, butit was always stable.DTMUV stimulation duck spleen and brain are reached the largest ratio increase on the first day. The spleen is 52.3 times, the brain is 371 times and it’s regulating rules similar to NDRV.This study has shown the two IRFs(IRF1 and IRF2) in the cherry valley duck, widely distributed in the tissues of healthy duck. After infected with the different viral, the expression of IRF1 and IRF2 in brain and spleen were up-regulated, suggesting that the two IRFs were involved in the antiviral response. This work laid a foundation for further research on the mechanism of the antiviral action of IRF1 and IRF2, which will help for further understand the innate immune system of the duck.