猪α-干扰素在毕赤酵母中的分泌表达及其生物活性测定

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

利用基因工程技术,将编码梅山猪α-干扰素成熟蛋白基因(mPoIFNα,501 bp)亚克隆到含分泌信号肽序列的毕赤酵母表达载体pPIC9K中,构建成分泌型重组表达载体pPIC9K-mPoIFNα。用化学方法(LiCl)将线性化的mPoIFNα与ssDNA共转化入毕赤酵母菌株GS115,转化子经MD平板筛选和PCR鉴定后,得到的阳性菌株再以高浓度的G418筛选多拷贝重组子。该高拷贝菌株经1%甲醇连续诱导4 d,表达产物经SDS-PAGE和Western-blot检测,结果表明在毕赤酵母中猪α-干扰素获得分泌型表达,表达产物约为20 000,在GS115中的表达量约为40mg/L,占GS115表达的可分泌型总蛋白的40.1%。对表达产物进行理化分析发现,重组酵母菌表达的蛋白耐酸(pH2),对热(56℃)部分敏感,并能被特异性抗猪α-干扰素抗体中和而不与抗猪γ-干扰素抗体反应。细胞病变抑制法(CPE50)测定干扰素生物活性,试验结果表明rPolIFNα具有较高的抗病毒生物活性,在MDBK中的抗VSV比活性为8.0×106U/mg。

To highly express secreted porcine interferon-alpha （PoIFNα）, the signal peptide was excised from the interferon-alpha gene and the mature gene （mPoIFNα, 501bp） was cloned into the yeast-Escherichia shuttle vector pPICgK to construct secreting recombinant expressing plasmid of pPIC9K-mPoIFNα. The pPIC9K-mPoIFNα was linearized by Sal I and co-transformed with ssDNA into Pichia pastoris cells GS115 （defective with histidine） with LiCl. The transformants were selected with MD culture plate and identified by PCR. And then, the multicopy recombinant Pichia pastoris strain was selected by G418 resistance. The selected strain could specifically secret 20 000 mPoIFNα proteins which was demonstrated by SDS-PAGE and Western-blot. The mPoIFNα proteins were about 40.1 % of total secreted proteins with the concentration of about 40 mg/L. The results of physicochemical property of the secreted protein indicated that the recombinant protein was insensitive to acid （pH2）, partly sensitive to heat （56℃） ,and the antiviral activity could be neutralized by anti-PoIFNα serum and not by anti-PoIFNγ serum. The antiviral activity of mPoIFNα was tested by CPE50 method. The results showed that the rPoIFNα was of high antiviral activity,which was 8.0 × 10^6 U/mg against VSV in MDBK cells.