猪γ-干扰素在毕赤酵母中的分泌表达及其抗病毒作用

Secreting expression of porcine interferon-gamma in Pichia pastoris and its antiviral activity

为了获得高效分泌表达重组猪γ-干扰素(rPoIFNγ) ,将去除信号肽的编码梅山猪γ-干扰素成熟蛋白基因(mPoIFNγ)插入酵母-大肠杆菌穿梭载体pPIC 9K中,构建成分泌型重组表达载体pPIC 9K-mPoIFNγ。将线性化的pPIC9K-mPoIFNγ以化学方法(LiCl)转化入毕赤酵母菌株GS115(组氨酸缺陷型) ,转化子经MD平板筛选和PCR分析鉴定后,以G418(4 g/L)筛选到多拷贝菌株。SDS-PAGE和Western-blot检测结果表明,所获得的重组子能够分泌表达出17 000和23 000左右的mPoIFNγ特异蛋白,其表达量约为120 mg/L,占分泌型总蛋白的65 %;细胞病变抑制法(CPE50)测定干扰素生物活性,结果表明rPolIFNγ具有较高的抗病毒生物活性,在MDBK中的抗VSV比活性为1.67×106U/mg。

To highly express secreted porcine interferon-gamma （rPoIFNT）, the signal peptide was excised from the interferon- gamma gene and the mature gene （mPoIFNT） was cloned into the yeast-Escherichia shuttle vector pPIC 9K to construct secreting recombinant expressing plasmid of pPIC 9K-mPoIFNγ. The pPIC 9K-mPoIFNγ was linearized and transformed into Pichia pastoris cells GSll5 with LiCl.The GSll5 was defective with histidine （His） .The transformants were selected with MD culture plate and identified by PCR. And then, the multicopy recombinant Pichia pastoris strain was selected by G418 （4 g/L） resistance. The selected strain could specifically secret 17 000 and 23 000 mPoIFN7 proteins which was demonstrated by SDS-PAGE and Western-blot. The PoIFN7 proteins amount to about 65% of total secreted proteins with the concentration of 1：20 mg/L.The antiviral activity of rPoIFNγ was tested by CPE50 method. The results showed that the rPoIFNγ had high antiviral activity, which was 1.67 × 10^6 U/mg against VSV in MDBK cells.