NDV F基因真核表达质粒的构建及其与鸡IL-18基因联合免疫的初步研究

Construction of Eukaryotic Expression Plasmid Containing NDV F Gene and Adjuvant Effect of Chicken IL-18

为了探索鸡新城疫病毒(NDV)F蛋白的免疫原性及鸡白细胞介素-18(IL-18)的免疫增强作用,通过PCR和酶切方法将NDV F基因克隆至真核表达载体pcDNA3.1(+),构建真核表达质粒pcDNA-F。用质粒pcDNA-F单独或与鸡IL-18重组真核表达质粒pIL-18联合免疫14日龄SPF鸡。首免后每周采取外周血及外周抗凝血,应用ELISA和MTT法分别检测免疫鸡的体液免疫及细胞免疫水平。二免后第14 d用NDV强毒F48E9株进行攻击。结果表明,质粒pcDNA-F与pIL-18联合免疫显著提高鸡体的细胞免疫水平,并且提供了50%免疫保护,这为ND的防治开辟了新的途径。

To investigate the immunogenicity of Newcastle disease virus（NDV） F protein and the immunoenhancement activity of chicken interleukin-18（IL-18）,the recombinant plasmid pcDNA-F was constructed by cloning F gene encoding the protective antigen F of NDV into eukaryotic express vector pcDNA 3.1（＋） with using the methods of the PCR and restricted enzyme cutting.Fourteen-day-old SPF chickens were immunized by two intramuscular inoculations of plasmid pcDNA-F alone or together with a recombinant eukaryotic expression plasmid expressing chicken IL-18.After the first immunization,peripheral blood lymphocytes were detected by MTT assay and the sera were collected to examine antibodies of chickens each week by ELISA.The chickens were challenged with 1000 ELD50NDV F48E9 on the 14th day post the booster.These results showed that co-immunization of plasmid pcDNA-F and pIL-18 could significantly enhance cell-mediated immune response,and 50% of the chickens were protected from homologous NDV challenge,which provided a new way for control of ND.