【作者】 刘文强；

【导师】 赵宏坤；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2005， 博士

【摘要】 牛羊白细胞介素18(Interleukin-18,IL-18)及其受体在家畜养殖业中是一种具有广阔应用前景的新型免疫佐剂和免疫治疗剂,在比较免疫学研究上也具有一定意义。根据已发表的IL-18 成熟蛋白cDNA 序列保守区设计一对特异性引物,应用RT-PCR 技术从山羊肺泡巨噬细胞和LPS/ConA 活化的脾细胞中扩增到编码山羊IL-18 成熟蛋白基因,其大小为480bp。将该基因克隆到pMD18-T 载体中,经序列测定表明山羊IL-18 与奶牛和绵羊的IL-18 基因序列高度同源,而且Caspase-1酶切位点的氨基酸序列没有发生变异,决定IL-18 活性的两个关键氨基酸位点也没有发生变异。将该基因从重组pMD-gIL-18质粒中亚克隆到表达载体pET-32a(+)质粒中。将重组表达载体在大肠杆菌BL21(DE3)中以1mol/L IPTG 诱导4h 或者更长时间进行表达。经SDS-PAGE 试验检测到了gIL-18 与pET 载体融合表达的重组蛋白。以该重组蛋白与佐剂混合后,免疫小鼠制备多克隆血清,经Western-blotting 试验证实该重组蛋白具有免疫原性。经过包涵体变性、层析柱纯化和复性,证实该蛋白具有诱导MDBK 细胞分泌IFN-γ的生物学活性,这为gIL-18 的实际应用奠定了基础。以β-actin 为内参照,使用半定量RT-PCR 法对来源于山羊肺脏单核巨噬细胞、外周血单核细胞(PBMC)、脾单个核细胞、肝细胞、肾单个核细胞、大脑和心肌组织的IL-18 mRNA 的表达进行了测定。结果表明:不论是否加入刺激剂,肺泡巨噬细胞都可以持续的表达IL-18 mRNA,但是被LPS 活化的PBMC 只是微弱的表达IL-18 mRNA,肝和脾细胞经LPS活化后也可以检测到IL-18 mRNA的表达,其水平明显高于PBMC,其它细胞和组织没有检测到IL-18 mRNA。这种在不同细胞中表达的差异可能反映了其产生成熟IL-18 的能力。IL-18结合蛋白(IL-18BP)具有拮抗IL-18生物学活性的作用,可视为IL-18的拮抗剂。以往的研究发现正痘病毒属的几种病毒基因组都含有编码IL-18BP的基因序列。在羊痘病毒基因组序列中也可能有编码IL-18BP的同源基因。以羊痘病毒作为模板,设计一对特异性引物,经PCR扩增了gIL-18BP基因,将该基因克隆到表达载体pET32a(+)中,经IPTG诱导,在大肠杆菌BL21(DE3)中实现了融合表达。目的蛋白以包涵体形式存在。表达产物经过变性、复性和层析纯化后,具有体外抑制羊IL-18刺激MDBK细胞产生IFN-γ的活性。这为研究IL-18主导疾病发生机制及治疗提供了理论和实践依据。更多还原

【Abstract】 Interleukin-18 (IL-18), a novel discovered cytokine is considered to be an usful adjuvant and therapeutics in control of diseases in intensive herd industries. Such studies of IL-18 and its receptors are useful for the progress of immunology. We isolated and sequenced a 480bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pMD18-T vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle and sheep`s. Conserved amino acid sequence at the enzymed site of caspase-1 and two key amino acid about its bioactivity are also observed. The gIL-18 gene from the recombinant plasmid was sub-cloned into pET32a(+) vector. The recombinant plasmid pET-gIL-18 in E.Coli BL21(DE3) was introduced by 1mmol/L IPTG for 4h or more time for expression. The fusion protein of pET and gIL-18 was expressed. SDS-PAGE analysis suggests that the recombinant protein is about 38ku. The recombinant protein can induce IFN-γproduction in MDBK cells after denature、renature and purified. Western-blotting indicated recombinant gIL-18 showed well reaction to its specific multi-antibodies. IL-18 mRNA from cells and tissues of goat are detected by RT-PCR,in whichβ-actinis is as a inner-control. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in PBMC treated by activator. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues. Some virus genome of vaccinia subgenus in poxvirus encoded IL-18BP gene, which can block IL-18 activity but no sequence similarity to membrane IL-18 receptors. Virus genome in sheep pox subgenus also be induced having such homologous gene. A pair of primers spanned the ORF of goat IL-18BP gene stemming from sheep poxvirus genome were designed. We cloned this gene and expressed it in E.Coli with pET32a(+) plasmid. After being purified,the recombinant fusion protein can inhibit goat IL-18 induce IFN-γproduction in MDBK. We amplified bovine mature IL-18 gene from the spleen cells in a milch cow by RT-PCR更多还原