IBDV VP2/4/3-ChIL-2融合基因真核表达载体的构建及其在Vero细胞中的表达

构建含有传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)VP2/4/3和鸡白细胞介素2(Chicken Interleukin 2,ChIL-2)融合基因的真核表达载体pCI-VP2/4/3-IL-2、pCI-IL-2-VP2/4/3,并在Vero细胞中进行表达.应用重叠延伸剪切技术(splicing by overlapping extension),分别经3次PCR获得融合基因片段VP2/4/3-IL-2、IL-2-VP2/4/3,定向插入真核表达载体pCI中,获得重组质粒pCI-VP2/4/3-IL-2、pCI-IL-2-VP2/4/3,在脂质体介导下,分别转染Vero细胞.RT-PCR检测证实导入的外源基因在Vero细胞中得到了转录;间接免疫荧光试验(IFA)和Western-blot检测证实重组质粒在Vero细胞中正确表达了插入的外源基因编码的融合蛋白,且表达的融合蛋白具有免疫反应性.重组真核表达载体pCI-VP2/4/3-IL-2、pCI-IL-2-VP2/4/3的成功构建及其在Vero细胞中的有效表达,为进一步探讨ChIL-2作为分子免疫佐剂对IBDV DNA疫苗的特异性免疫增强作用奠定了基础.

Construction of an eukaryotic expression vector containing IBDV VP2/4/3 and ChIL-2 fusion gene and their expression in Vero cells

Recombinant eukaryotic expression vectors containing fusion genes of infectious bursal disease virus (IBDV) polyprotein (VP2/4/3) gene and chicken interleukin 2 (ChIL-2) gene were constructed using the technique of splicing by overlapping extension (SOEing) and three times PCR. Fusion gene fragments were directly cloned into pCI, an eukaryotic expression vector, to produce recombinant expression plasmids pCI-VP2/4/3-IL-2 and pCI-IL-2-VP2/4/3. The recombinant plasmids were transfered into Vero cells introduced by Lipofectamine2000 reagent. Gene specific RT-PCR showed that the fusion genes were successfully transcribed in Vero cells. The fusion proteins were detected by indirect immunofluorescence and western-blot assays. These results suggest that the fusion genes were expressed correctly in Vero cells, thus providing a basis for immune-enhancement of ChIL-2 in DNA vaccination against IBDV.