大熊猫GM-CSF基因的克隆与分析及GM-CSF生物学活性的检测  

Cloning and analysis of GM-CSF gene from giant panda and detection of biological activities of GM-CSF

作　　者：李凤琴[1] 徐志文[1] 朱玲[1] 古锋[1] 王承东[2] 李德生[2] 郭万柱[1] 

机构地区：[1]四川农业大学动物医学院动物生物技术中心,四川雅安625014 [2]中国保护大熊猫研究中心雅安碧峰峡基地,四川雅安625014

出　　处：《中国兽医科学》2012年第10期1063-1067,共5页Chinese Veterinary Science

基　　金：卧龙大熊猫俱乐部基金项目(201001);四川省青年科技基金项目(2012JQ0037)

摘　　要：用含刀豆蛋白的营养液培养大熊猫外周血中分离的淋巴细胞,72h后抽提RNA。设计1对引物,通过RT-PCR方法对大熊猫GM-CSF基因片段进行扩增。测序后对其进行分析,并将该序列克隆到pCI-neo真核载体中,命名为pCI-G,然后免疫健康小鼠,同时设立pCI-neo载体和PBS免疫组为对照。序列分析结果显示,该基因片段含一个完整的开放性阅读框,编码144个氨基酸,与其他几个物种同源性为76.1%～86.2%,预测蛋白中含较多的α-螺旋区域,主要集中在3个区域。免疫结果显示,与对照组相比,pCI-G能够引起小鼠体内IL-4和IFN-γ持续快速的增长,在首免后第56天,二者的质量浓度可达到70.86pg/mL±4.22pg/mL和237.39pg/mL±3.17pg/mL。该质粒可增强小鼠Th2型和Th1型免疫应答,是极具应用潜力的细胞因子。Lymphocytes originated from peripheral blood of giant pandas were cultured by enchyma with ConA,and the RNA was prepared.GM-CSF gene in giant pandas was amplified with RT-PCR,analysed and cloned into pCI-neo after being sequenced.Healthy mice was immunized with the recombinant plasmid pCI-G by intramuscular injection,and the pCI-neo vector and PBS immunization group were used as control.The gene contained a complete open reading frame and encoded a 144 amino acid homology.Homology was from 76.1% to 86.2% among several other species.The protein encoded by GM-CSF gene in giant panda contains more α-helix region,mainly concentrated in three regions.Compared with the mouse in control group,the contents of IL-4 and IFN-γ in the mouse in pCI-G group had a sustained and rapid growth.On day 56 after the first injection,the contents of IL-1 and IFN-γ were 70.86 pg/mL±4.22 pg/mL and 237.39 pg/mL±3.17 pg/mL,respectively.The results showed that the plasmid could enhance mice of Th2 and Th1 immune response,and was a potential cytokine.

关 键 词：大熊猫 GM-CSF基因 生物学活性 白细胞介素4 Γ-干扰素 

分 类 号：S852.42[农业科学—基础兽医学]