γ-干扰素原核高效表达体系的建立

Construction of an efficient prokaryotic expression system for producing γ-IFN protein

用基因重组的方法，将γ－ＩＦＮｃＤＮＡ 片段用ＥｃｏＲＩ单酶插入到原核表达载体ｐＢＶ２２０ 中，筛选得到带有正向串联了２个γＩＦＮｃＤＮＡ 片段的阳性重组质粒，转化大肠杆菌ＤＨ５α后，建立了γ－ ＩＦＮ 的原核表达体系。经温度诱导培养，ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎ－ ｂｌｏｔ、ＳＤＳ－ ＰＡＧＥ 光密度扫描检测证实：该体系可高效表达γ－ ＩＦＮ 蛋白，表达量占菌体可溶性蛋白的４０ ％以上。病变抑制法测活表明：表达产物具有γ－ ＩＦＮ 的抗病毒活性。这一体系的建立为大规模发酵生产γ－ ＩＦＮ 提供了可靠的工程菌株，为进一步研究其分离纯化工艺提供了重要的实验模型。

The plasmid pIFN γ has been constructed by the insertion of γ-IFN cDNA into pBV220,RE mapping showed that the structure of pIFNγ agreed with this design. Then the plasmid pIFN γ was transformed into E. Coli DH5α to establish the prokaryotic expression system for γ-IFN protein. SDS-PAGE, Western-blot confirmed that the system can express efficiently γ-IFN protein, which was over 40% of the total bacterial soluble protein.γ-IFN antiviral activity has been determined by virus inhibition test. The establishment of this system provides an efficient expression cell lines for producing γ-IFN protein on a large scale.