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| 鸡白细胞介素18全长基因的克隆、表达及分子进化分析 | |
| 引用本文： | 许健,于涟,李龙,由振强,万旺军,刘岩.鸡白细胞介素18全长基因的克隆、表达及分子进化分析[J].科技通报,2007,23(1):52-57,101. |
| 作者姓名： | 许健 于涟 李龙 由振强 万旺军 刘岩 |
| 作者单位： | 1. 浙江大学,生物医学工程和仪器科学学院,杭州,310027;新疆石河子大学,医学院,新疆,石河子,832002 2. 浙江大学,动物预防医学研究所,浙江省重点实验室,杭州,310029 3. 浙江大学,生物医学工程和仪器科学学院,杭州,310027 |
| 基金项目： | 国家高技术研究发展计划(863计划);浙江省科技计划;浙江省自然科学基金 |
| 摘 要： | 根据鸡白细胞介素18（IL-18）cDNA基因序列设计了1对特异性引物，应用RT-PCR技术，从脂多糖（lipopolysaccharide，LPS）刺激的我国地方品种萧山鸡原代鸡脾细胞中扩增并克隆鸡IL-18全长基因（Genbank accession，AY628648）。扩增片段全长591bp，共编码197个氨基酸的前体蛋白，其中含有表达完整功能蛋白所必需的起始密码子和终止密码子。该序列与国外报道的鸡IL-18全长基因核苷酸序列及推导的氨基酸序列的同源性为98．99％。只在引导序列中出现两个氨基酸残基的缺失，分子进化分析表明萧山鸡IL-18基因与火鸡以及家鸭基因形成一个独立的分支。将萧山鸡IL-18成熟蛋白序列插入pGEX-4T-2载体并在Ecoli中得到表达，获得45kD的GST-IL-18融合蛋白。经纯化后用于制备多克隆抗体。本研究对萧山鸡白细胞介素18的全长基因进行了克隆及表达，并对其分子进化进行了分析。为深入研究其功能奠定了基础。 |
| 关 键 词： | 鸡白细胞介素18 克隆 序列分析 分子进化 |
| 文章编号： | 1001-7119（2007）01-0052-06 |
| 收稿时间： | 2005-06-15 |
| 修稿时间： | 2005-06-15 |
Cloning, Expression and Phyiogenetic Analysis of Interleukin-18(IL-18) Gene in Xiaoshan Chickens |
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| XU Jian,YU Lian,LI Long,YOU Zhen-qiang,WAN Wang-jun,LIU Yan.Cloning, Expression and Phyiogenetic Analysis of Interleukin-18(IL-18) Gene in Xiaoshan Chickens[J].Bulletin of Science and Technology,2007,23(1):52-57,101. | |
| Authors: | XU Jian YU Lian LI Long YOU Zhen-qiang WAN Wang-jun LIU Yan |
| Institution: | 1. Collage of Biomedical Engineering and instrument science, Zhejiang University, Hangzhou 310027,China; 2. Institute of Preventive Veterinary Medicine,Zhejiang provincial key laboratory, Hangzhou 310029, China; 3. Medical College of Shihezi University, Xinjiang Shihezi 832002, China |
| Abstract: | The ChIL-18 mRNA was isolated from chicken splenocytes 10h after stimulated with LPS.ChIL-18 gene was amplified from chicken primordial spleen cells induced by lipopolysaccharide(LPS) with RT-PCR(Genbank accession,AY628648).The RT-PCR products were inserted into prokaryotic(pGEX-4T-2) expression vectors.One 591 bp cDNA fragment,containing complete open reading frame(ORF) of ChIL-18 gene,was obtained in RT-PCR.The deduced ChIL-18 amino acid(aa) sequence of Xiaoshan chicken is the same as that from HD-11 cell except 2aa difference.Phylogenetic analysis showed that the avian IL-18s together,apart from the mammalian sequences,separated with a long branch between them.Recombinant ChIL-18 protein was obtained in prokaryotic expression system,and its polyclonal antibody was performed in rabbits.We obtained ChIL-18 gene from the activated splenocytes of Xiaoshan chicken.The recombinant ChIL-18 protein expressed in E.coli strain BL21 has immunoreactivity and immunogenicity.These results gave a basis for further research of the bioactivity of ChIL-18.To further understand the evolution of chicken interleukin-18(IL-18),and also with the eventual aim of using cytokine to influence the immune response to pathogens in chicken. |
| Keywords: | interleukin-18 clone sequence analysis phylogenetic analysis |
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