摘要
为了高效表达分泌型的猪β-干扰素,将去除信号肽的编码梅山猪β-干扰素成熟多肽基因（mPoIFNβ）插入酵母-大肠杆菌穿梭载体pPIC9K,构建成分泌型重组表达载体pPIC9K-mPoIFNβ。将以SalⅠ线性化的pPIC9K-mPoIFNβ用化学方法（LiCl）,与鲑鱼精DNA（ssDNA）共转化入毕赤酵母菌株GS115,转化子经MD平板筛选和PCR鉴定后获得阳性菌株,再经高浓度G418筛选到多拷贝菌株。以1%甲醇连续诱导4 d,SDS-PAGE和Western blot检测结果表明：表达产物为22 ku和26 ku的混合物,在GS115中的表达量约为80 mg/L,占分泌型总蛋白的29.4%-30.8%,表明猪β-干扰素基因在毕赤酵母中获得了高效分泌表达。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,并能被特异性的抗猪β-干扰素抗体中和而不与抗猪γ-干扰素抗体反应。以细胞病变抑制法（CPE50）测定干扰素抗病毒活性,在MDBK（牛肾细胞系）中表达的猪β-干扰素的抗VSV比活性达到2.0×10^6U/mg;对口蹄疫病毒在IBRS-2细胞中的增殖也有明显的抑制作用。
To highly express secreted porcine interferon-β （rPoIFNβ）, the signal peptide was excised from the porcine interferon-β gene and the mature gene （mPoIFβ） was cloned into the yeast-Escherichia shuttle vector pPIC9K to construct secreting recombinant expressing plasmid of pPIC9K-mPoIFNβ. The pPIC9K-mPoIFNβ was linearized by Sal Ⅰ and co-transformed with ssD NA into Pichia pastoris cells GS115 with LiCl. The GS115 was defective with histidine （His）. The transformants were selected with MD culture plate and identified by PCR. And then, the muhicopy recombinant Pichia pastoris strain was selected by G418 resistance. The selected strain could specifically secret 22 ku and 26 ku mPoIFNβ proteins which were demonstrated by SDS-PAGE and Western blot. The mPoIFNβ proteins were about 29.4 %-30.8 % of total secreted proteins and the concentration was about 80 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-PolFNβ serum but not by anti-PolFNy serum. The antiviral activity of rPoIFNβ was tested by CPE50 method. The results showed that the rPoIFNβ was of high antiviral activity, which was 2.0×10^6 U/mg against VSV in MDBK cells. The rPoIFNβ could suppress propagation of FMDV in IBRS-2 cells.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2007年第5期506-512,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家863计划基金(2002AA245071)
湖北省科技攻关(2004AA202B01)资助项目
