鸡γ-干扰素的可溶性表达及纯化产物的抗NDV活性

将鸡γ-干扰素(chIFN-γ)基因先克隆,接着通过大肠杆菌表达后进行产物纯化与活性检测.通过PCR方法以带有信号肽的重组质粒PMD-18T-preIFN-γ为模板,扩增无信号肤chIFN-γ基因片段,克隆至原核表达载体pProEXTM HTa,构建重组表达质粒pProEXTM HTa-chIFN-γ,在大肠杆菌BL2...

Soluble expression of chicken interferon-gamma and activity of purified expression product against NDV

chIFN-γ mature protein gene was amplified by RT-PCR from recombinant PMD-18TpreIFN-γ and then cloned into the prokaryotic expression vector pProEXTMHTa.Recombinant expression plasmid of pProEXTMHTa-chIFN-γ was constructed and then transformed into the competent E.coli BL21(DE3) cells for expression with IPTG induction.Purified soluble rchIFN-γ proteins were obtained by His.Bind Purification Kit(Novagen) and identified by SDS-PAGE gel and Western blot assay.The antiviral activity was detected by CEF-NDV system.A 459 bp cDNA encoding chIFN-γ mature protein gene was cloned and successfully expressed in E.coli with approximate molecular weight of 20 ku,which could be recognized by anti-His mAb against chIFN-γ.The recombinant chIFN-γ proteins were expressed to form inclusion bodies with a portion of soluble protein.The soluble rchIFN-γ proteins were purified by Ni-NTA column under a native condition with the yield of 1.9 mg.mL-1.The purified recombinant chIFN-γ(rchIFN-γ) proteins by 1∶54dilution could resist 100 TCID50 NDV virus attack.There fore,the purified rchIFN-γ proteins by Ni-NTA column under a native condition had better antiviral activity,which will establish a basis for further developing new type antiviral interferon praeparatum.