摘要
以RT PCR方法 ,从经丝裂原诱导的猪外周血淋巴细胞总RNA中扩增出编码猪IFNγ的基因。经测序证实后插入载体pJLA 5 0 3 ,并实现在大肠杆菌中的高表达。表达产物以包涵体形式存在 ,经 7mol/L盐酸胍变性及精氨酸存在的情况下复性。再经DEAE Sepharose离子交换柱、Sephdex 2 0 0凝胶过滤柱分离获得电泳单一纯猪IFN γ蛋白 ,细胞病变抑制实验检测纯化产物有干扰素活性。
Peripheral blood lymphocytes from a single swine were stimulated with Concavadin A for 17h,and the total RNA was isolated from it.Then,the mRNA specific for porcine IFNγ was amplified by reverse transcription polymerase chain reaction.After sequencing,the IFNγ gene has been successfully inserted into vector pJLA\|503 and highly expressed in E.coli. Recombinant porcine IFNγ expressed as inclusion body,which was dissolved in 7mol/L guanidine chloride and subsequently renatured by dilution in refolding buffer containing 0.5mol/L L\|arginine.In order to obtain pure protein,the renatured IFNγ was pruified by the chromatographies of SP\|Sepharose FF and Sephacryl S\|200 HR.As a result,the final pure product can been seen as a single band in SDS\|PAGE, and the cytokine activity was verified by inhibiting the cytopathic effect.
出处
《生物工程学报》
CAS
CSCD
北大核心
2001年第2期183-186,共4页
Chinese Journal of Biotechnology
基金
国家高技术研究发展计划项目! (10 1 0 6 0 5 0 4)
关键词
Γ干扰素
基因表达
分离纯化
大肠杆菌
interferon\|γ,gene expression,isolation and purification, E.coli
