梅花鹿γ-干扰素在不同真核细胞系中稳定表达的研究  

Stably Expressing Research of Interferon-gamma (IFN-γ) from Cervus Nippon in Different Eukaryotic Cell Lines

作　　者：刘颖[1,2] 王冰[1,2] 于清龙[1,2] 熊家军[3] 杨利国[3] 陈焕春[1,2] 郭爱珍[1,2] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]华中农业大学动物医学院预防兽医学省重点实验室,武汉430070 [3]华中农业大学动物科技学院,武汉430070

出　　处：《中国畜牧兽医》2010年第5期85-89,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家973项目(2006CB504401);湖北省自然基金重大项目(2008CDA073);艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2009ZX10602-14;2008ZX10301)

摘　　要：提取经植物血凝素诱导培养的梅花鹿外周血淋巴细胞总RNA,应用RT-PCR方法扩增出梅花鹿γ-干扰素成熟蛋白基因,经克隆测序表明与GenBank上发表的干扰素序列同源性为100%。将其重组到含有CMV增强子的真核表达载体质粒pCI-neo上。利用磷酸钙介导转染法将重组载体质粒pCI-neo-CerIFN-γ转染入中国仓鼠肾细胞(BHK-21)和牛肾细胞(MDBK)中,在G418抗性压力下进行筛选培养获得了稳定分泌表达的转染细胞系。通过Western blotting检测确定表达产物的相对分子质量分别为23、20、16ku,与预测大小一致。本研究成果为进一步开发梅花鹿生物制品类治疗制剂奠定了基础。Total RNA was isolated from Cervus Nippon peripheral blood lymphocytes,which were stimulated with PHA. Then the Cervus Nippon mature IFN-γ gene was amplified by reverse transcription chain reaction. The results indicated that the cloned gene was mature Cervus Nippon IFN-γ gene,which had the identities of 100% with the CerIFN-γ gene published in the GenBank,subcloned into the eukaryotic expression plasmid pCI-neo vector which had the CMV enhancer. The recombinant pCI-neo-CerIFN-γ plasmids were transfected into BHK-21 cell and MDBK cell by calcium phosphate. The stably expression of transfected cell lines were screened by the G418 resistance. Detected by Western blotting,to determine the relative molecular expression products were 23,20,16 ku,it is consistent with the forecast size. This research results lay the foundation for further develop of the Cervus Nippon biological preparations class treatment.

关 键 词：梅花鹿 Γ-干扰素 细胞系 真核表达 

分 类 号：Q78[生物学—分子生物学]