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591 bp,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E.coli中得到表达,获得45 kD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。
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 prokaryotie 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 immunoreaetivity 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 eytokine to influence the immune response to pathogens in chicken.
出处
《科技通报》
2007年第1期52-57,101,共7页
Bulletin of Science and Technology
基金
国家"863"高科技研究发展计划资助(101-j99-02)
浙江省重点科技项目(011102465)
浙江省自然科学基金重大项目(No.ZA0105)
