裂谷热病毒囊膜蛋白基因DNA免疫的研究

分别将裂谷热病毒(Rift Valley Fever Virus,RVFV)囊膜糖蛋白GN、GC和G(N C)基因亚克隆至真核表达载体pCAGGS多克隆位点鸡β-actin转录启动子下游,分别构成pCAGG-RVFV-GN、pCAGG-RVFV-GC和pCAGG-RVFV-G(N C)。免疫沉淀试验结果表明,重组RVFV蛋白GN、GC分别在pCAGG-RVFV-GN、pCAGG-RVFV-G(N C)转染HeLa细胞中获得表达,并具有良好免疫反应性。pCAGG-RVFV-GN、pCAGG-RVFV-GC和pCAGG-RVFV-G(N C)质粒DNA混合物按100μg/只剂量肌肉注射免疫6周龄BALB/c小鼠。每隔4周用相同的剂量加强免疫,第二次加强免疫3周后采血、分离血清备用。分别以杆状病毒表达RVFV囊膜蛋白GN、GC制备的抗原液包被ELISA板,间接ELISA检测DNA免疫鼠血清中RVFV囊膜蛋白G(N C)特异性抗体,具有良好的敏感性和特异性。另外,DNA免疫鼠血清中的特异抗体可有效中和RVFV囊膜蛋白G(N C)介导的伪型VSV重组病毒侵入RVFV易感宿主细胞的感染性。结果表明,pCAGG-RVFV-GN、pCAGG-RVFV-GC和pCAGG-RVFV-G(N C)质粒DNA混合物作为DNA疫苗具有防制裂谷热的潜力。

Study on DNA immune of envelope protein gene of Rift Valley Fever Virus

DNA vaccines have successfully induced effective antibody and cellular immune response to many viral pathogens. The antibody response of DNA immunization induction in mouse model with envelope glycoproteins of Rift Valley Fever Virus (RVFV), G (N + C), GN and GC was investigated. For this purpose, three codon G (N + C), GN and GC gene were insert into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C). The expression of recommbinant GN or / and GC protein in BHK cells transfected with pCAGG-RVFV-GC or pCAGG-RVFV-G (N + C) DNA were confirmed by immunoprecipitation. Six-week-old female BALB/c mice were intramuscularly primed with 100 (g pCAGG-RVFV-GN + pCAGG-RVFV-GC + pCAGG-RVFV-G (N + C), and boosted with same dose after 4 weeks. The serums were collected at 3 weeks post final boost. The serum IgG against Rift Valley Fever Virus G (N + C) protein were detect by indirect ELISA using recombinant Baculovirus expressed Rift Valley Fever Virus GN and GC glycoprotein. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C) elicited much strong IgG response. For serum neutralization antibody assay, a recombinant Vesicular Stomatitis Virus pseudotype, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVdeltaG x G, Whitt M A) and complemented with Rift Valley Fever Virus G (N + C) glycoprotein expressed in transient (VSVdeltaG x RVFV-G), was use to replace the authentic Rift Valley Fever Virus. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C) also induced high titer of neutralization antibody response. These result indicates that DNA immunization is an efficient vaccine strategy against Rift Valley Fever Virus.