三黄鸡α干扰素的克隆、表达及抗病毒活性初步研究

Molecular Cloning,Expression and Antiviral Activity of Three-Yellow Chicken IFN-α

采用PCR方法从三黄鸡血液基因组中扩增鸡α干扰素全基因,并克隆和测序。序列分析表明,基因全长为582bp,亚克隆其成熟蛋白编码基因489bp。将该片段与表达载体pET-28a连接克隆至大肠埃希菌DH5α菌株,经测序和酶切鉴定,选取正向插入、读码框正确的阳性克隆。构建重组质粒并转化BL21(DE3),经IPTG诱导,融合蛋白以包涵体形式在BL21(DE3)高效表达,经His亲和层析柱得以纯化。鸡胚试验表明,重组干扰素抵抗新城疫病毒效果显著,能保护鸡胚并能够延迟和减少新城疫病毒的复制。

The full length of chicken interferon alpha gene was amplified by polymerase chain reaction from the genome DNA of Three-Yellow chicken blood,and then sequenced.The amplified gene was 582 bp.The coding region（489 bp）for mature protein was subcloned into the prokaryotic expression vector pET-28a.The recombinant vector was cloned into Escherichia coli DH5α strain.The positive clone gene with correct reading frame was confirmed by sequencing and enzyme digestion.The results of SDS-PAGE revealed that the recombinant plasmid that was transformed into BL21（DE3） was highly expressed after induction by IPTG and the 22 ku fusion protein was expressed in the form of inclusion bodies.The recombinant protein was purified with His-Bind affinity chromatography.Chick embryo tests showed that recombinant chicken interferon alpha can protect chick embryo from NDV infection.Recombinant chicken interferon alpha can also delay and reduce the NDV replication significantly.