摘要
将ILTVgB基因和鸡IL-18基因插入到真核双表达载体pIRES中,构建共表达gB和IL-18基因的重组质粒pIRES-gB/IL18和表达gB基因的pIRES-gB质粒。通过脂质体将其转染鸡胚成纤维细胞,利用RT-PCR及间接免疫荧光检测重组质粒在体外的表达。将重组质粒通过腿部肌肉多点注射免疫21日龄雏鸡,应用ELISA和流式细胞仪分别检测免疫鸡的体液免疫及细胞免疫水平。结果显示,pIRES-gB/IL18和pIRES-gB转染细胞中分别含gB/IL-18基因的mRNA和gB基因的mRNA。间接免疫荧光检测表明,pIRES-gB/IL18和pIRES-gB在CEF细胞中有效地转录并表达gB蛋白。pIRES-gB/IL18和pIRES-gB免疫雏鸡后均能促进外周血T淋巴细胞亚群数量和血清中特异性抗体水平的增加,但前者的免疫效果明显优于后者,表明gB基因和鸡IL-18基因均获得了表达,且鸡IL-18能明显增强ILTV DNA疫苗诱发的免疫应答。
The research was to construct eukaryotic coexpression vector of avian infectious laryn- gotracheitis virus （ILTV） gB and chicken IL-18, and its immunogenicity was evaluated. The gB gene of ILTV and chicken IL-18 gene were inserted into the bicistronic plasmid pIRES to con- struct recombinant plasmids pIRES-gB and plRES-gB/IL18. Plasmids were transfected into the chicken embryo fibroblasts （CEF） cells by using lipofectamine, respectively, and the mRNA was detected by RT-PCR and the expressed products were detected by indirect immunofluorescence assay. The 21-day-old chickens were immunized intramuscularly with plasmids, and the induced humoral and cellular responses were evaluated, gB mRNA expression was found in CEF cells and gB protein expression in CEF cells was detected by the indirect immunofluorescence test. Immunization with pIRES-gB/IL18 led to a more dramatic augmentation of humoral and T cell responses than pIRES-gB which demonstrated that ILTV gB and cIL-18 gene were expressed, and IL-18 could significantly enhance the immune response of ILTV DNA vaccine.
出处
《四川农业大学学报》
CSCD
北大核心
2011年第4期549-554,共6页
Journal of Sichuan Agricultural University
基金
国家"十一五"科技支撑计划专项(2006BAD06A08)
