Tandem-expressing of Newcastle disease virus F and Chicken Interleukin-2 (IL-2) Genes in Eukaryotic Expression Vector and Their Immunogenicity | 鸡新城疫病毒F基因与鸡白介素-2(IL-2)基因在真核载体中串连表达及免疫原性

Chinese. 为了探索鸡白介素-2(IL-2)对鸡新城疫病毒(NDV)F蛋白免疫效果的增强作用，本研究通过重叠延伸PCR技术(SOE-PCR)利用连接子(G2SG3S)将鸡IL-2基因和鸡NDV F基因串连，克隆至真核表达载体pcDNA3.1(+)，构建了表达质粒pcDNA-IL-2-F。通过脂质体介导DNA瞬时转染法，将重组质粒pcDNA-IL-2-F和pcDNA-F分别转染鸡胚成纤维细胞，Western blot验证表明，pcDNA-IL-2-F和pcDNA-F均能瞬时表达F蛋白，表达蛋白分别为76.0和59.6 kD，表达蛋白的大小与预期一致并具有抗原性。将表达质粒免疫SPF鸡(Gallus domedticus)，6 d后开始用ELISA方法检测NDV的特异抗体，免疫20 d后，用F48E9进行攻毒。结果显示，免疫pcDNA-IL-2-F的鸡群产生的抗体水平(P0.05)高于混合免疫pcDNA-IL-2和pcDNA-F，高于免疫pcDNA-F的鸡群，但比免疫Lasota组的抗体水平要低。攻毒试验说明免疫pcDNA-IL-2-F组比免疫其他质粒组保护率高，这说明串连表达的免疫效果要好于二者的联合使用，为病毒病疫苗的研制提供了思路。

English. To investigate immuno-enhancement activity of chicken interleukin-2 (IL-2) to Newcastle disease virus (NDV) F protein, NDV F gene was linked with chicken IL-2 via 7-amino acid glycine-rich linker(G2SG3S) by SOE-PCR(splicing by overlap extension-PCR) technique and then inserted into pcDNA3.1(+) and constructed the recombinant plasmid pcDNA-IL2-F which was transfected into chicken embryo fibroblast cells with liposome. The recombinant poteins were expressed correctly with molecular mass of 76.0 kD and 59.6 kD. The results of Western blotting analysis indicated that the interest proteins(pcDNA-IL-2-F and pcDNA-F) were recognized specifically by antisera against NDV. SPF chickens(Gallus domedticus), and were divided into 5 groups and immunized by recombinant plasmid pcDNA-IL-2-F, pcDNA-IL-2 mixing pcDNA-F, pcDNA-F, Lasota vaccine and plasmid pcDNA-3.1(+), respectively. The sera of each group were collected weekly for detecting antibodies of immunized chickens by ELISA. The results suggested that SPF chickens immunized pcDNA-IL-2-F exhibited stronger antibody response(P0.05) than that of both the group immunized mixing pcDNA-F with pcDNA-IL-2 and the group immuned pcDNA-F. The SPF chickens were challenged with NDV F48E9 on the 20th day post immunization and the protective rate of the group immunized pcDNA-IL-2-F was higher than that of other plasmid groups. These results revealed that expression of tandem genes of NDV F and Chicken IL-2 could provide an optional immune response that in turn protected chickens from NDV challenge than that of sigle NDV F gene. In general, the vaccine strategy described in this study can provide a good platform for future desire of virus vaccine.