猪干扰素α、β和γ的融合表达及活性

Study on the Fusion Expression and Bioactivity of Porcine Interferon α,β and γ

【目的】研制高效的猪基因工程干扰素制剂用于猪病毒性和肿瘤性疾病的防治。【方法】采用重叠延伸PCR(splicing by overlap extension PCR,SOE-PCR)技术将PoIFN-α、PoIFN-β和PoIFN-γ成熟肽基因进行连接,形成3种融合基因PoIFN-α/β、PoIFN-α/γ和PoIFN-β/γ,并构建到原核表达载体pET30a进行融合表达,用细胞病变抑制试验和淋巴细胞转化试验测定融合蛋白rPoIFN-α/β、rPoIFN-α/γ和rPoIFN-β/γ的生物学活性,同时与非融合干扰素rPoIFN-α、rPoIFN-β和rPoIFN-γ进行比较分析。【结果】成功构建了3种融合基因,并在大肠杆菌中实现表达,经纯化、复性得到3种具有生物学活性的融合蛋白rPoIFN-α/β、rPoIFN-α/γ和rPoIFN-β/γ。细胞病变抑制试验结果表明,无论是在Marc-145还是在PK-15细胞上,3种融合蛋白均具有较强的抗病毒活性,其抗PRRSV或VSV活性明显高于非融合的rPoIFN-α、rPoIFN-β和rPoIFN-γ,体现出一定的叠加效应;MTT法检测淋巴细胞转化试验结果显示,rPoIFN-α/γ和rPoIFN-β/γ能有效刺激淋巴细胞转化,具有良好的免疫学活性;而rPoIFN-α/β效果不明显。说明Ⅰ型与Ⅱ型PoIFN之间能协同调节免疫,而Ⅰ型的不同亚型之间无明显协同作用。【结论】3种融合干扰素具有较强的抗病毒活性,可以用于猪病毒性和肿瘤性疾病的防治,其中rPoIFN-α/γ和rPoIFN-β/γ还具有较强的免疫增强作用,可被开发成新型的免疫增强剂。

[Objective] The research aimed to exploit high efficient porcine gene engineering antiviral agents to prevent and control the porcine viral diseases and tumor diseases. [Method] Three fusion genes of PolFN-α/β, PolFN-α/γ and PoIFN-β/γ, were constructed by linking any two mature peptide genes of the PolFN-α, PolFN-β and PolFN-γ by SOE-PCR method. Then, these fusion genes were subcloned into prokaryotic expression vector pET-30a for expression in E. coli_BL21（DE3）. Subsequently, the bioactivities of the fusion proteins rPoIFN-α/β, rPoIFN-α/γ and rPoIFN-β/γ were comparatively analyzed with non-fusion proteins rPoIFN-α, rPolFN-β and rPoIFN-γ by the cytopathic effect （CPE） inhibition assay and the porcine lymphocyte transformation assay. [Result] The fusion genes of PolFN-α/β, PoIFN-α/γ and PolFN-β/γ/were successfully constructed and expressed in E. coll. After purified and refolded from inclusion bodies, three fusion proteins of rPolFN-α/β, rPoIFN-α/γ and rPolFN-β/γ with well biological activity were obtained successfully. The results of the cytopathic effect （CPE） inhibition assay indicated that all these three kinds of fusion proteins, which demonstrated more significant antiviral activities for PRRSV or VSV than non-fusion proteins rPolFN-α, rPoIFN-β and rPolFN-y not only on Marc-145 but also on PK-15, exhibited increased antiviral potencies. The effects of rPoIFN-α/β, rPolFN-α/y and rPolFN-β/y on the porcine lymphocyte transformation were detected by the MTT method. The results suggested that both rPolFN-α/7 and rPoIFN-β/y could effectively stimulate the transformation of porcine lymphocyte, had obvious immunoregulatory activities, but rPolFN-α/β had no obvious effect. So, there are synergistic immunoregulatory effects between type I and type II PolFNs, and none between PolFNs of type I. [Conclusion] All these three fusion PolNFs in study, which exhibited the prominent abilities of antivirus, could be used to prevent and control the porcine viral diseases and tumor diseases. And the rPolFN-α/γ and rPolFN-β/γ also had stronger immune enhancement and could be expected to exploit into new-type immunopotentiators.