Cloning Of Chicken TMEM9B Homologous Gene&Immune Function Study Of Akirin2Gene

In recent years, with the continuous emergence of new infectious diseases, the studyfields of vaccine prevention and immune mechanisms become one of the focuses again.Also, TMEM9B and AKIRIN2were recently found, which play important roles in theimmune signaling pathways and inflammatory responses. Further studying the functionsand characters of these immune-related new genes, it has important theoretical andpractical reference value for deeply understanding of the immune defense mechanismsand developing new biological agents for disease control. Chicken with its uniquebiological characteristics, becomes one of important model animals for immunologyresearch. In this study, chicken is used as experimental animal, and we cloned Tmem9Bgene and preliminarily studied the immune functions of akirin2gene. The main researchmethods and the experimental results are as follows:Tmem9B is an important and indispensable factor of Tumor Necrosis Factor (TNF)in the activation of nuclear factor-kappaB (NF-κB) and Mitogen-activated protein kinase(MAPK) pathways, and is involved in the formation of membrane lipid rafts, and cellsignaling and the regulation of NF-κB. In order to clone this gene in chicken, wepredicted the full length of Hi-Line Brown chicken Tmem9B cDNA contig sequenceusing in silico cloning. Specific primers were designed basis on this predicted sequence,so the sequence was cloned using RT-PCR technique. Then the sequence was cloned intothe pMD18-T vector for sequencing. We did elements prediction and structures analysisabout Tmem9B homologous nucleotide and amino acid sequences using bioinformaticsmethods, as well as did sequence homology comparison analysis and evolutionaryanalysis about the different species Tmem9B homologous genes.Various tissues andinternal organs at different developmental stages of chicken were used for RNAextraction, and tissue expression profile and identification of tmem9B homologous genewere analyzed using semi-quantitative RT-PCR technique. Tmem9B homologous genewas subcloned into the expression vector pcDNA3.1to construct the recombinantplasmid pcDNA3.1-tmem9B for following research. The results show that: We succeedto obtain Hi-Line Brown chicken Tmem9B homologous gene full-length coding sequenceof570bp, and these results are consistent with the predictions. Bioinformatics analysis shows that there are three kinds of modification sites (myristoylation sites, glycosylationsites and protein kinase C phosphorylation sites), a signal peptide in Tmem9B amino acidsequence, and Tmem9B may be a glycoprotein which is located in the lysosomal and theearly endosomal transmembrane. Tmem9B gene expression activity has differences indifferent tissues and different developmental stages of chickens, which shows that thechicken Tmem9B may be an immune-related protein that has variety of functions.Akirin2is one of the important strict nuclear regulator, which plays key roles in thetranscriptional regulation of NF-κB dependent genes. In this study, the chicken akirin2gene full-length coding sequence was subcloned to construct the recombinant eukaryoticexpression vector pcDNA3.1-akirin2, then, the plasmid pcDNA3.1-akirin2was largelyextracted and purified. The non-vaccinated Hi-Line Brown chickens were vaccinatedwith Newcastle disease LaSota vaccine. At the same time, the pectoral muscles of thesechickens were injected with the plasmid pcDNA3.1-akirin2, and the control groupchicken were injected with pcDNA3.1-EGFP plasmid. After inoculation, we drewchicken blood serum randomly every week, which were used for detection and analysisserum antibody titers using Hemagglutination Inhibition (HI) Test. The results show that:pcDNA3.1-akirin2can significantly enhance the level of antibodies against the LaSotavaccine after immunization, whereas the control groups that injected withpcDNA3.1-EGFP have no impact on the antibody levels. This study will establishpreliminary experimental foundation for the possible application of the recombinantplasmid pcDNA3.1-akirin2used as genetic engineering immunostimulants in veterinaryclinical.Through the above of the chicken Tmem9B homologous gene cloning andexpression characteristic identification, and akirin2gene immune function study, thesebasic researches will not only establish the foundation for further studying these genesfunctions, but also provide the references for the function study of human relatedhomologous genes.