摘要
通过RT-PCR技术直接从罗曼鸡脾脏提取的总RNA中扩增鸡白细胞介素18(ChIL-18)全基因,并克隆和测序得到序列基因全长为597 bp的ChIL-18。将ChIL-18成熟蛋白编码区(510 bp)定向插入到原核表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)基因的下游,构建重组表达质粒pGEX-ChIL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下可高效表达融合蛋白(GST-ChIL-18)。SDS-PAGE可检测到分子质量约为46 kDa的GST-ChIL-18。Western blot证实GST-ChIL-18能与鸡IL-18单克隆抗体发生特异性反应。融合蛋白GST-ChIL-18经谷胱苷肽Seph-arose-4B亲和柱层析纯化后明显促进鸡T淋巴细胞转化,表明所表达的ChIL-18具有一定的生物活性。
Chicken interleukin-18（ChIL-18） gene was amplified from total RNA extracted directionally from Luoman chicken splenocytes by reverse transcription-polymerase chain reaction（RT-PCR）.PCR product was cloned into pGEM-T Easy vector and sequenced.The result showed that the nucleotide sequence of chicken IL-18 gene was 597 bp,including the stop coden and the same as the published chicken IL-18 cDNA sequence by Schneider K.A prokaryotic expression plasmid of chicken IL-18 gene,pGEX-ChIL-18,was obtained by subcloning the encoding region of chicken IL-18 mature peptide gene into pGEX-4T-1. The recombinant chicken IL-18 was expressed efficiently in pGEX-ChIL-18-transformed BL21 （DE3） LysS induced by IPTG, the yield accounted for 18.6%of the total bacterial protein. The results showed that fusion protein GST-ChIL-18 was about 46 kDa and could react with monoclonal antibody for chicken IL-18. After purified by Glutathione Sepharose-4B affinity column, chicken IL-18 protein induced obvious proliferation of chicken splenocytes in vitro.
出处
《安徽农业大学学报》
CAS
CSCD
北大核心
2009年第4期547-551,共5页
Journal of Anhui Agricultural University
基金
国家"十一五"科技支撑计划专项(2006BAD06A08)资助
关键词
鸡白细胞介素18
成熟蛋白
克隆
原核表达
chicken interleukin-18
mature protein
cloning
prokaryotic expression
