大熊猫秦岭亚种IL-2基因的克隆表达及其生物活性研究  

Study on the cloning,in vitro expression and bioactivity of Ailuropoda melanoleuca Qinling subspecies interleukin-2

作　　者：乔琦[1] 马清义 朱辉[1] 陈德坤[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]陕西野生动物抢救中心,陕西西安710400

出　　处：《细胞与分子免疫学杂志》2009年第6期479-482,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家林业局大熊猫国际合作项目(WH0632)

摘　　要：目的:明确大熊猫四川种群和秦岭种群IL-2在基因水平是否存在差异;得到具有具有生物活性的秦岭大熊猫IL-2原核表达产物。方法:采用RT-PCR方法从ConA诱导培养的秦岭大熊猫外周血淋巴细胞总RNA中扩增得到IL-2的cDNA片段,利用DNA重组技术构建原核表达载体pET32a-IL-2,转入BL21(DE3)中,以IPTGBL21(DE3)表达IL-2融合蛋白。融合蛋白进行SDS-PAGE和Western blot进行鉴定及可溶性分析;用纯化的融合蛋白免疫家兔,制备多克隆抗体;采用淋巴细胞增殖试验测定融合蛋白生物活性。结果:通过重组表达获得IL-2抗原蛋白,相对分子质量(Mr)为34000;免疫家兔得到特异性的抗IL-2抗体,该抗体能够检测细胞内源性IL-2蛋白的表达;融合蛋白具有促进淋巴细胞转化增殖的生物学活性,并且它的这种作用可被本研究制备的多抗所抑制。结论:成功克隆了秦岭大熊猫IL-2基因,与四川大熊猫核苷酸同源性为99.4%表达了具有生物活性的融合蛋白。AIM：To identify if any difference exisit between the population of Sichuan and Qinling giant panda. express IL-2 protein in BL21（DE3）with biological activity. METHODS：IL-2 was amplified by RT-PCR from adult Qinling giant panda peripheral blood lymphocyte which was induced by ConA,and cloned into prokaryotic expression vector of pET32a-IL-2. The fusion protein was expressed in BL21（DE3）/pET system and identified by SDS-PAGE and Western blot; inoculate rabbit with purified expression products to prepare polyclonal antibody; use lymphocytes proliferation in vitro to detect biological activity. RESULTS：The protein of IL-2 was obtained by recombination expression,molecule weight is 34 000.The specificity of polyclonal antibody was obtained,in vitro activity test indicated that the recombinant protein IL-2 having an activity of promoting the proliferation of lymphocytes. The effect can be stopped by polyclonal antibody which was prepared before. CONCLUSION：The IL-2 gene of Qinling giant panda was cloned and expressed in E.coli successfully,and the homology of IL-2 gene in these two population is 99.4% and the recombination protein can promote the proliferation of lymphocytes in vitro from giant panda.

关 键 词：秦岭大熊猫 IL-2 克隆 多克隆抗体 生物活性 

分 类 号：S858.9[农业科学—临床兽医学]