鸡粒细胞-巨噬细胞集落刺激因子的克隆与真核表达  

Cloning and eukaryotic expression of chicken granulocyte macrophage colony stimulating factor gene

作　　者：谭兵[1] 王红宁[1,2] 张毅[2] 樊汶樵[2] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川大学生命科学学院动物疾病防控与食品安全四川省重点实验室,四川成都610064

出　　处：《浙江大学学报（农业与生命科学版）》2009年第5期516-522,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家高技术研究发展计划863"资助项目(2006AA10A205)

摘　　要：为了研究鸡粒细胞-巨噬细胞集落刺激因子(GM-CSF)的生物学功能,采用反转录-聚合酶链式反应(RT-PCR)技术从刀豆蛋白(ConA)刺激的四川山地乌骨鸡外周血淋巴细胞中扩增出GM-CSF基因,进一步通过PCR等方法将该基因亚克隆入真核表达载体pCI-neo,构建出重组表达质粒pCI-GM-CSF,将pCI-GM-CSF转染COS-7细胞,提取表达产物进行活性检测.序列测定表明,四川山地乌骨鸡GM-CSF全基因长为435 bp,编码144个氨基酸,与GenBank上公布的瓦赫宁鸡GM-CSF基因(AJ621740)氨基酸序列同源性达95.1%,与其他哺乳动物GM-CSF基因的氨基酸序列同源性低于30%.骨髓细胞增殖实验证实四川山地乌骨鸡GM-CSF能明显促进鸡骨髓细胞的增殖.以上结果表明,本实验成功克隆了四川山地乌骨鸡GM-CSF基因,证实四川山地乌骨鸡GM-CSF重组蛋白具有明显的生物学活性.In order to study the biological characteristics of chicken granulocyte macrophage colony stimulating factor（GM-CSF）, Sichuan shandi black-bone chicken GM-CSF was amplified from ConA stimulated peripheral blood lymphocytes using RT PCR. The chicken GM-CSF cDNA was subcloned into the eukaryotic expression plasmid pCI-neo to construct recombinant p｜asmid pCI-GM-CSF. Then, the plasmid pCI-GM-CSF was transfected into COS-7 cell. The bioactivity of expressed GM-CSF protein was measured. The result shows that Sichuan shandi black-bone chicken GM-CSF gene had 435 bp in length and encoded 144 amino acids. Its sequence had 95.1~~ identity with that of Wageningen chicken GM-CSF gene published in GenBank （accession No. AJ621740）, and less than 30% identity with those of mammalian at the amino acid level. The assay of proliferation of chicken bone marrow cell confirmed that Sichuan shandi black-bone chicken GM-CSF could enhance bone marrow cell proliferation obviously. The result above indicates that Sichuan shandi black-bone chicken GM-CSF is cloned successfully and has obviously biological activity.

关 键 词：粒细胞-巨噬细胞集落刺激因子 四川山地乌骨鸡 真核表达 生物学活性 

分 类 号：Q78[生物学—分子生物学]