摘要
应用RT-PCR技术分别从脂多糖(LPS)活化的牛脾脏细胞和肺泡巨噬细胞中克隆到编码牛白细胞介素(bIL-)18成熟蛋白的cDNA基因,其大小为480 bp。将bIL-18克隆到pET32a(+)质粒中,转化大肠杆菌BL21,经IPTG诱导后进行SDS-PAGE分析,得到了分子量为38ku的融合蛋白,该蛋白经纯化并复性后,具有诱导MDBK分泌IFN-γ的活性,并且可以刺激ConA活化的外周血单核细胞(PBMC)增殖。使用半定量RT-PCR对其mRNA的体内表达进行了测定,发现不论是否加入刺激剂,牛巨噬细胞都可以持续的表达IL-18 mRNA,但是被LPS活化的PBMC只有微弱表达,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,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。
We isolated and sequenced a 480 bp cDNA encoding mature bovine interleukin-18 （bIL-18） from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The bIL-18 gene was cloned into pET32a （＋） vectors and sequenced. Nucleotide sequence of bIL-18 shares high homology with cattle. Fusional expression with pET32a （＋） of bIL-18 of about 38ku was obtained by SDS-PAGE analysis after induction by IPTG in the E. coli BL21 expression system. The recombinant protein can augment T cell proliferation ConA-stimulated and IFN-7 production by MDBK. The IL-18 mRNA was constitutively detected in bovine 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 peripheral blood mononuclear cells （PBMCs） treated by activators. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2005年第9期873-876,共4页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家"十五"乳业重大科技专项(2002BA518A16)
山东省"三O"工程基金(2003_3009)
