以伴刀豆球蛋白A(ConA)诱导的犬外周血淋巴细胞中提取的总RNA为模板,通过RT PCR的方法克隆扩增出犬α干扰素基因,将所扩增基因克隆于原核表达载体pBV220 并进行测序,结果显示该基因与GenBank上所公布的犬α干扰素基因同源性为100%。将重组表达载体转入宿主菌进行温敏诱导表达,表达产物经SDS PAGE分析,证明目的蛋白以包涵体的形式存在,大小约为19kDa。将表达产物变性、复性、透析、纯化处理后加入犬肾细胞上,用水泡性口炎病毒攻毒,测出重组的CaIFN α具有较高的抗病毒作用,生物活性达到5.11×106 ∪/ ,重组蛋白质的含量约为13 /L。

With the reverse transcription chain reaction (RT-PCR), the gene of Canine′s interfe ron-alpha (CaIFN-α) was amplified and cloned , from the total RNA in the lymphocytes stimulated with concanavalin A from the peripheral blood of dog. The amplified fragment was inserted into the prokaryotic expressing vector pBV220 and then sequenced. Sequencing result showed that the homology was 100% between the gene we cloned and the one published in GenBank. SDS-PAGE assay showed that the target protein, with the molecular weight of 19kDa, could be expressed in a high level and up to 27.6% of the total protein in E.coli cell, in the form of inclusion bodies. The target protein was added to MDCK cells,which were subsequently infected by the 100 TCID_(50) VSV. The result showed good cytokine activity. The recombinant CaIFN-α activity was up to 5.11×10~6U/㎎.