CHO细胞表达重组猪干扰素-γ及其抗病毒活性

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity

摘要:

为利用中国仓鼠卵巢(Chinese hamster ovarian, CHO)细胞表达系统制备重组猪干扰素-γ (recombinant porcine interferon gamma, rPoIFN-γ)，并分析其体外抗病毒活性，本研究首先构建rPoIFN-γ真核表达质粒pcDNA3.1-PoIFN-γ，转染悬浮培养的CHO细胞，进行上清分泌表达，利用亲和层析纯化目的蛋白，并进行SDS-PAGE和Western blotting鉴定；通过CCK-8实验分析rPoIFN-γ对细胞的毒性，用VSV/PK-15系统检测其抗病毒活性效价；最后分析rPoIFN-γ对塞内卡病毒A (Seneca virus A, SVA)的抗病毒活性及其对细胞内干扰素刺激基因(interferon-stimulated genes, ISGs)和细胞因子的诱导作用。结果显示，本研究利用CHO悬浮细胞表达系统成功制备了纯化的rPoIFN-γ，该rPoIFN-γ对细胞无毒性，VSV/PK-15系统检测其活性效价为5.59×10⁷ U/mg；此外，rPoIFN-γ可诱导细胞内多种ISGs和细胞因子的表达，并显著抑制SVA的复制。总之，本研究成功利用CHO表达系统制备了高活性的rPoIFN-γ，并在体外完成其对SVA的抗病毒作用分析，为rPoIFN-γ的功能及抗病毒制剂的研究提供了实验材料和基础。

Abstract:

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×10⁷ U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.