鸡α干扰素基因的原核表达及其活性测定

Prokaryotic expression and activity assay of recombinant chicken interferon-alpha

应用RT-PCR技术,从被刺激诱导的鸡脾脏淋巴细胞中扩增鸡α-干扰素(ChIFN-α)基因cDNA,经克隆和测序后,构建原核表达重组质粒pGEX-proChIFN-α(全长序列)和pET-ChIFN-α(不含信号肽序列),并分别转化相应的表达菌。重组菌经IPTG诱导,SDS-PAGE结果表明,ChIFN-α基因均获得表达,重组蛋白大小分别为45ku、26ku左右,表达的蛋白以包涵体形式存在。表达产物经变性、提纯、复性,重组蛋白的含量约为1mg/mL。复性后的ChIFN-α能够在鸡胚成纤维细胞上抑制H5N1禽流感病毒的复制;用水泡性口炎病毒检测结果表明,重组ChIFN-α具有较高的抗病毒作用,其生物学活性达到2×106u/mg。

Chicken IFN-α gene was amplified by RT-PCR and cloned from chicken spleen lymphocytes stimulated by concanavalin A. Recombinant plasmids （pGEX-proChIFN-α, pET-ChIFN-α ） were expressed in E. coli. SDS-PAGE analysis showed that the expression products had a molecular weight of 45 ku and 25 ku respectively, and existed in inclusion bodies. The expressed protein was shown to inhibit H5N1 avian influenza virus or vesicular stomatitis virus replication on chick embryo fibroblast （CEF） at 2 × 10^6 u/mg. The potential of chicken IFN-αas adjuvant was discussed.