摘要
为获得鸭α-干扰素(DuIFN-α)并研究其生物学功能,将DuIFN-α成熟蛋白基因与原核表达载体pET32a+连接后转化到BL21(DE3)获得工程菌BL2l(DE3)/pET32a+-DuIFN-α,对其表达产物的纯化和复性关键技术及其抗水疱性口炎病毒(VSV)、鸭瘟强毒(DPV)活性进行研究。结果表明:BL2l(DE3)/pET32a+-DuIFN-α经0.4mmol/L IPTG诱导获得高效表达,表达的重组蛋白相对分子质量约37 ku,以包涵体形式存在;重组蛋白经镍金属螯合介质(Ni-MIAC)进行纯化和复性后获得理想效果,其抗VSV活性的比活力达到12 800 U/mg;利用定量PCR检测到15 U/mL的重组DuIFN-α对鸭瘟强毒表现出抑制作用,为重组DuIFN-α的临床应用提供试验数据。
In order to obtain DulFN a and investigate its biological function, duck interferon-α （DuIFN-α） mature protein gene was cloned in to pMD18-T vector and inserted into the expressing plasmid/ pET32a＋. Then, this recombinant plasmid was transformed into engineering bacteria/ BL21（DE3） and a 37 kD protein was expressed in the form of inclusion bodies after 5 hours induction by 0.4 mmol/L IPTG. The expressed product which was purified and refolded by metal immobilization affinity chromatography（MIAC） have an ideal activity against virus. Its activity of anti-VSV in DEF was up to 12 800 U/rag and DuIFN-α at 15 U/mL can reduce duck plague virus replication by 80% as measured by FQ-PCR.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2008年第12期1753-1758,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家"十一五"科技支撑计划(2007Z06-017)
教育部科技创新工程重大项目培育资金项目(7060)
教育部"新世纪优秀人才支持计划"项目(NCET-04-0906/NCET-06-0818)
四川省教育厅重点项目(2003A024)
四川省重点建设学科项目(SZD0418)
关键词
鸭IFN-α
成熟肽基因
原核表达
色谱复性
抗病毒活性
duck IFN-α
mature peptide gene
prokaryotic expression
purification refolding bychromatography
activity against virus
