鸡IFN-γ基因在大肠杆菌中的表达及表达产物对鸡的免疫保护

Expression of chicken IFN-γ gene in Escherichia coli and immunological protection of the expressed protein for chickens

将鸡干扰素-γ(ChIFN-γ)成熟肽基因编码区序列亚克隆到原核表达载体pQE30上,并转化大肠杆菌M15感受态细胞。经诱导后,以镍金属亲和层析法对表达蛋白进行纯化,采用SDS-PAGE与Western-blot对表达蛋白进行鉴定。结果显示,经诱导的转化子在体外获得了高效表达,表达蛋白约占菌体蛋白的23%。0.5ng/mL以上的重组ChIFN-γ(rChIFN-γ)能显著抑制鸡新城疫病毒对细胞的侵染与伤害;但是,细胞先感染病毒后,再以不同剂量rChIFN-γ作用于细胞,则不具有对细胞的保护作用,说明rChIFN-γ对细胞的保护是通过调节机体免疫能力、干扰病毒入侵实现的。1ng/mL的rChIFN-γ可显著提高新城疫疫苗的免疫效价,具有较强的免疫协同作用。表明,原核表达的rChIFN-γ具有显著的生物学活性。

The mature peptide coding sequence of chicken IFN-γ（ChIFN-γ） gene was cloned into pQE30 vector and then transformed into Escherichia coli M15. The recombinant with ChIFN-γ gene was expressed with induction of IPTG and the expressed protein was identified by SDS PAGE and Western blotting using 6 × His-tag monoclonal antibody. Under denaturing conditions, the expressed rChlFN-γ was purified using the immobilized metal affinity chromatography technology with Ni NTA agarose,and refollittle protection for CEFs challenged with NDV,indicating that the main mechanism of rChIFN -γ against NDV was to regulate the immunological ability and to interfer with virus invasion. In addition, 1 ng/mL rChIFN-γ could improve significantly the antibody level of the NDV vaccine, and the SPF chickens were protected against NDV invasion. These results revealed that the expressed rChIFN-γ had significant biological activity.