萧山鸡IL-2基因真核表达载体构建及其在Vero细胞中的表达

扩增萧山鸡白细胞介素2(IL-2)基因,将其插入真核表达载体pCI构建重组质粒pCI-IL-2,高压电击法将重组质粒转化入减毒沙门氏菌,并直接转染Vero细胞,提取细胞总DNA,DIG标记探针可检测到阳性杂交信号.用FITC标记的羊抗兔IgG进行间接免疫荧光试验,可检测到特异性黄绿色荧光.SDS-PAGE和Western blotting可检测到18.5 kD和14.5 kD的蛋白条带.表明减毒沙门氏菌能将重组质粒pCI-IL-2携带进入Vero细胞内表达IL-2,且表达产物具有免疫反应性,为今后研制鸡IL-2基因作为口服免疫增强剂提供了依据.

Construction of eukaryotic expression vector of Interleukin-2 gene from Xiaoshan Chicken and its expression in Vero cells

In previous study IL-2 gene of Xiaoshan Chicken has been cloned by our laboratory, and in this study the cDNA of IL-2 was amplified and inserted into the eukaryotic expression vector pCI. The resulted recombinant plasmid pCI-IL-2 was transformed by electroporation into an attenuated Salmonella typhimurium strain ZJ111, which was then used to transfect Vero cells. DNA extracted from the transfected Vero cells showed a positive signal as hybridized with DIG-labeled probe in a DNA blotting assay, which revealed that the IL-2 gene was integrated into the genome of Vero cells. Specific yellow-green fluorescence was visual in indirect immunofluorescence assay with FITC-labeled goat anti-rabbit IgG. The 14.5 kD and 18.5 kD bands correspond to IL-2 protein from lysate of Vero cells was identified by SDS-PAGE and Western blotting, indicating that in Vero cells IL-2 was expressed successfully with the characteristics of immunological reactivity.