【作者】 何江帅；

【导师】 卢强；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 硕士

【摘要】 本研究应用核酸杂交法,以鲤鱼白细胞介素-1βcDNA部分片段为探针,筛选丝裂原刺激的鲤鱼外周血白细胞cDNA文库,克隆了白细胞介素-1β全长cDNA序列,测得全长序列为1213bp,包含831bp的开放阅读框,推测编码276个氨基酸：根据所获得的全长cDNA序列设计引物,以鲤鱼脾脏基因组DNA为模板,克隆了白细胞介素-1β基因组DNA,序列全长为2451bp,其中含有6个内含子和7个外显子,并且外显子和内含子剪接位点都符合GT-AG规则；荧光定量PCR分析鲤鱼正常白细胞和有丝分裂原刺激的白细胞中,白细胞介素-1β基因不同时间段以及不同刺激物作用后的转录差异,实验结果表明：有丝分裂原刺激的白细胞中白细胞介素-1β转录增强,特别是LPS、PHA联合刺激后转录增强极显著,但是随着时间的延长转录增强的幅度呈下降趋势,可见白细胞介素-1β的转录对不同的诱导物敏感度不同,推测白细胞受刺激早期发挥免疫作用。更多还原

【Abstract】 At present,the higher animals have been on the role of cytokines and their insight into the mechanism, and some cytokines can be used in clinical treatment.such as interferon.interleukin and tumor necrosis factor. In recent years.non-mammalian vertebrate cytokines research is becoming increasingly important factor.especially in the economic value of aquatic animals cytokines.Cytokines are a class of biologically active substances.which are secreted by immune cells and non-immune cells. As signal transduction molecules between cells can be antigen antibodies and other information to the antigen receptor to mediate inflammation;As signal transduction molecules between cells.The antigen information can be passed to the antibodies and other antigen receptor to mediate inflammatory response;At the same time.they also regulate the immune response.stimulate of hematopoietic function and participate in tissue repair and so on. Interleukin-1(3 is a potential humoral immune response activator, which plays a key role in the local and systemic inflammatory response. It is generated mainly by blood monocytes and tissue macrophages. Interleukin-1βcan promote lymphocyte,macrophage, NK cell activation. also is a chemical inducer of neutrophil and mononuclear cells and involved in TH2 immune response.Interleukin-1βas a potential inflammatory mediators and costimulatory signal molecules,can increase macrophage phagocytosis.induce fever and promote the synthesis of prostaglandins.regulate the expression of other cytokines.As an immune regulatory cytokine, it has the potential to enhance the immune response.In this study, we obtained the nucleic acid probe with carp IL-1βcDNA fragments of by fluorescence mRNA differential display.After digoxigenin labeled. application of nucleic acid hybridization screening mitogen stimulated peripheral blood leukocyte cDNA library of carp, we acquire carp IL-1(3 cDNA sequence.According to sequence non-translated region and exon of IL-1βcDNA the pair of primers were designed.IL-1βgenomic DNA sequences was obtained by the PCR.Finally,on the basis of the IL-1βcDNA and genomic DNA sequences the pair of primers were designed.by Real time quantitative PCR method, analysis of isolated and cultured in the normal and mitogen stimulation leucocytes in the transcription of the gene difference between the situation at different times. The results showed that: we get the carp IL-1βcDNA clone full-length 1213bp. containing the 5’ and 3’ untranslated region and 3’ poly (A) polyadenylation signal (AATAAA), Open reading frame size of 831bp, encoding 276 amino acids,the homology of the deduced protein sequence to Carassius auratus.zebrafish.mice,cattle,human IL-1βwere 71%.54%, 22%,24% and 24%. Genomic DNA length is 2451 bp,sequence and phylogenetic analysis,the gene evolutionary is conservation, as with most species contain 7 exons and 6 introns,number of bases of each exon is close to mammalian exons correspondingly,exon and intron splice sites are complied with GT-AG rules. Quantitative PCR analysis showed that Mitogen-stimulated leukocytes increased transcription of IL-1β,especially the enhanced result of joint stimulation from LPS and PHA is particularly significant, but gradually decreased in stimulated leucocytes as the stimulation time going.It suggests IL-1βtranscription is different sensitivities to different inducers,also shows that the leucocytes play a immune role by early stimulating.更多还原