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当前位置: 首页 > 文章 > 鸡白细胞介素18基因的克隆及其在大肠杆菌中的表达 安徽农业大学学报 2009,36 (4) 33-37
Position: Home > Articles > Gene cloning and mature protein expression of chicken interleukin-18 in Escherichia coli Journal of Anhui Agricultural University 2009,36 (4) 33-37

鸡白细胞介素18基因的克隆及其在大肠杆菌中的表达

作  者：
李新生;尹红征;陈红英;邵攀峰;王振亚;宋亚鹏
单  位：
河南农业大学信息与管理科学学院;河南农业大学牧医工程学院
关键词：
鸡白细胞介素18;成熟蛋白;克隆;原核表达
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。
译  名：
Gene cloning and mature protein expression of chicken interleukin-18 in Escherichia coli
作  者：
LI Xin-sheng1,YIN Hong-zheng2,CHEN Hong-ying1,SHAO Pan-feng1,WANG Zheng-ya1,SONG Ya-peng1 (1.College of Animal Husbandry and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002;2.College of Information and Management Science,Henan Agricultural University,Zhengzhou 450002)
关键词：
chicken interleukin-18;mature protein;cloning;prokaryotic expression
摘  要：
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.

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