摘要
用RT-PCR从经ConA刺激的马外周血单个核细胞(PBMC)中扩增出马白细胞介素18(Equine interleu-kin-18,EIL-18)前体蛋白(precursor EIL-18,pEIL-18)基因的cDNA,然后克隆至载体pCR2.1-TOPO中,鉴定并命名为pCR2.1-pEIL-18。自pCR2.1-pEIL-18中扩增马白细胞介素18成熟蛋白(mature EIL-18,mEIL-18)基因,并将其亚克隆至原核表达载体pET-28a(+)中。将筛选出的阳性克隆进行测序、诱导表达并纯化其表达产物。结果表明,mEIL-18基因全长474 bp,含1个开放阅读框,编码157个氨基酸的成熟蛋白;表达产物以可溶性和包涵体两种形式存在,经SDS-PAGE和Western-blot分析,重组蛋白相对分子量约为20 ku,且具有免疫生物学活性。mEIL-18基因在大肠杆菌中高效表达并在非变性和变性条件下采用Ni+亲和层析纯化方法获得了高纯度的重组mEIL-18,为探究马白细胞介素18的生物学活性及其应用奠定了基础。
Using the total RNA extracted from ConA stimulated equine peripheral blood mononuclear cells （PBMC） as template, the cDNA of interleukin 18 was amplified by RT-PCR. The cDNA was subsequently cloned into the vector pCR2.1-TOPO and sequenced. Then the gene encoding mature equine interleukin 18 was amplified from the recombinant plasmid named pCR2. 1- pEIL-18 by polymerase chain reaction （PCR） and subcloned into pET-28a（＋）. The recombinant plasmid pET-mEIL-18 was transformed into E. coli BL21 （DE3） after sequence analysis, and a fusion protein was then expressed and purified. Results showed that the fragment of mEIL-18 gene was 474 bp in length, which contained an open reading frame and encoded the protein of 157 aa, and the expressed product existed in soluble portion and inclusion bodies. The SDS-PAGE and Western-blotting analysis indicated that the fusion protein was 20 ku in molecular weight and had immunological activity. The mEIL-18 was expressed successfully in E. coli BL21 （DE3）, and purified efficiently using Ni^＋ column chromatography in non-denatured and denatured conditions, which lays the foundation for investigating the structure and biological effects of EIL-18.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2007年第3期307-312,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
黑龙江省自然科学基金项目(ZJN-0602-01)
