| The natural immune system recognizes the invaded pathogen-associated molecular patterns (PAMPs) through the expression of different pattern recognition receptors PRRs and immune defence against all kinds of pathogen microorganisms by the cascade signal transduction. Melanoma differentiation associated gene MDA5 and NALP3 are important members of the Nucleotide-binding oligomerization domain-like receptors (RLR) family and Nucleotide-binding oligomerization domain-like receptors (NLR) sub family, respectively. They can identify the RNA virus, and regulate cytokine, exert the effect on non specific antiviral. Avian leukosis virus subgroup J (Avian leucosis virus subgroup J, ALV-J) is one of the avian leukaemia that has been widely concerned in recent years. The chickens which have been infected by this kind of virus often occur in immune suppression and will resulting in the blocking of immune. In order to understand the roles of MALP3 and MDA5 in fiber cells infected by ALV-J, one strain of ALV-J was isolated and identified,, and then establishes a method to infection induced by MALP3 and MDA5 mRNA transcription level detection of ALV-J virus by real-time fluorescence quantitative RT-PCR.Firstly, our laboratory isolated and identified 1 strains of ALV-J from suspected chickens in one layer farm in Hefei, Anhui province. Chicken with depression was thin, pale skin. After the autopsy, the liver, the spleen and the ovary swelled obviously. There were a large number of white tumor nodules on the surface of the liver. We adopted the tissue grinding, filtering method. Put the liver disease liquid inoculated chicken embryo fibroblast cell and DF-1 cell line, and then extracted and cultured 7d cells of total RNA and DNA. H5/H7 specific primers and env gene primers were amplified by PCR using, got 545bp and 2106bp size of the band respectively. Inserted the two fragments into pMD-18T vector, and sequenced positive clone from the transformation of E.coli DH5 alpha. The results showed that the laboratory successfully separated clinical isolates strains from a variety of tumors, named AJV-HF211. Homo logy analysis by DNA Man software in 14 strains of ALV-J which have been reported at home and abroad showed that the homology of AJV-HF211 strains and 14 strains is 88.8%-94.0%. AJV-HF211 has the closest relationship with the Guangdong isolates GD1109 is 94.0%, the homology is highest, the phylogenetic relationship is nearest and homology with the British original strain HPRS-103 is 89.3% only. They are not on the same branch, and the phylogenetic relationship is relatively far. It shows that AJV-HF211 has great variation in the process of genetic evolution.Secondly, this study designed the specific primer of NALP3 and MDA5 receptor, and established the method of SYBR Green real-time fluorescence quantitative detection of NALP3 and MDA5 receptor transcription level, and detected the transcription levels of two receptors mRNA in different period of DF-1 cells infected with AJV-HF211 strain in this method. Results show that, compared with the uninfected control cells, the NALP3 and MDA5 gene of AJV-HF211 strain infected DF-1 cells transcriptional levels had increased in varying degrees. NALP3 gradually rose in the amount of lst-3rd day after infection (DAI) expression after virus infection, reached the peak at 4th DAI, and fluctuated at from 5th to7th DAI. The NALP3 expression had a sharp decline after 5th DAI. Moreover, the MDA5 in the group of virus infection was slowly rising at l-3d, decreased at the 4th DAI, and increased at 5th-6th DAI, expression and reached the highest at 6th DAI, and decreased at 7th DAI. Our results suggested that MDA5 and NALP3 receptors play an important role in the process of ALV-J infection, and the related mechanism need further verification. |
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