摘要
提取经ConA诱导培养的猪外周血白细胞总RNA ,RT -PCR扩增出猪IFN -γ基因并克隆到pGEM -T -easy载体 ,测序结果表明扩增片段是含有信号肽的猪IFN -γ的完整基因。设计 1对引物亚克隆猪IFN -γ成熟蛋白基因并对其5′端前 2个稀有密码子进行大肠杆菌偏嗜性改造。经测序鉴定后插入原核表达载体pRLC ,并实现在大肠杆菌中的高效表达。表达产物以包涵体形式存在 ,经 7mol·L-1盐酸胍的变性液溶解及 0 5mol·L-1盐酸胍复性液复性处理 ,表达产物进行脱盐、凝胶层析纯化。细胞病变抑制法测定结果表明 ,重组猪IFN -γ具有较高的干扰素活性 ,约为 4 5× 10 6U·mg-1。
Total RNA was isolated from swine peripheral blood lymphocytes which were stimulated with Concavadin A.Then the porcine IFNγ cDNA was amplified by reverse transcription polymerase chain reaction.The amplified fragment was cloned into vector pGEMTeasy and then sequenced.The result indicates that the cloned gene is a complete porcine INFγ gene with the codes for signal peptide.Another pair of primers was designed to sub clone the gene coding porcine IFNγ mature protein and the two rare codes near 5′ terminal were modified to the hobby of E coli .After sequencing,the sub cloned IFNγ gene was successfully inserted to expression vector pRLC and highly expressed in E coli .Recombinant porcine IFNγ expressed as inclusion body,which was dissolved in 7 mol·L -1 guanidine chloride and subsequently renatured by dilution in refolding buffer containing 0 5 mol·L -1 guanidine chloride.In order to obtain pure protein,the renatured poIFNγ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S200 chromatography.As a result,the purified product was verified to be of high cytokine activity by inhibiting the cytopathic effect.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2003年第2期71-75,共5页
Journal of Nanjing Agricultural University
关键词
猪Γ干扰素
基因克隆
基因改造
基因表达
活性
细胞病变抑制法
porcine interferon gamma(poIFNγ)
gene modification
prokaryotic expression
isolation and purification
