摘要
提取经 Con A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 p GEM- T- easy载体 ,测序结果表明 ,扩增片段为含有信号肽的猪 IFN-γ完整基因。亚克隆猪 IFN-γ成熟蛋白编码基因 ,并对其 5′段前 2个稀有密码子进行大肠杆菌偏嗜性改造。通过引入 SD序列 ,成功构建了猪 IFN- γ原核双顺反子表达载体 p L CS-po IFN2 G,并实现了猪 IFN-γ在大肠杆菌中的高表达 ,约占菌体总蛋白的 5 6 .5 %。表达产物以包涵体形式存在 ,用含 7mol/L 盐酸胍的变性液溶解及含 0 .5 mol/L盐酸胍复性液复性处理 ,复性后的表达产物经凝胶层析纯化后 ,细胞病变抑制法测定结果表明 ,重组猪
Total RNA was isolated from swine peripheral blood lymphocytes which were stimulated with Concavadin A.Then the mRNA specific for porcine IFN-γ was amplified by reverse transcription polymerase chain reaction.The sequencing result indicates that the cloned gene is a complete porcine IFN-γ gene with the codes for signal peptide.Two pairs of primers were designed to sub-clone the gene coding porcine IFN-γ mature protein and to introduce the ribosome binding side core gene to another porcine IFN-γ mature protein gene.The two rare codes near 5′ terminal of porcine IFN-γ mature protein gene were mutant to the usage of E.coli.Porcine IFN-γ bicistron expression vector pLCS-poIFN2G were successfully constructed by using the two sub-cloned genes.Recombinant porcine IFN-γ was highly expressed in E.coli,which accouts for 56.5 percent of the total protein of the ferment genetic E.coli.Recombinant porcine IFN-γ expressed as inclusion body,which was dissolved in 7 mol/L guanidine chloride and subsequently renatured by dilution in refolding buffer containing 0.5 mol/L guanidine chloride.In order to obtain pure protein,the renatured poIFN-γ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography.As a result,the purified product was verified to be of high cytokine activity by inhibition test of the cytopathic effect.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2004年第1期52-55,共4页
Chinese Journal of Veterinary Science
