尼帕病毒融合蛋白和受体结合蛋白基因DNA免疫的研究

构建了表达哺乳动物密码子优化的NiV囊膜蛋白F和G基因的真核表达质粒pCAGG-NiV-F和pCAGG-NiV-G.细胞融合试验表明,重组NiV融合蛋白F和受体结合蛋白G在pCAGG-NiV-F、pCAGG-NiV-G共转染BHK细胞中获得表达,并具有良好生物学活性.真核表达质粒pCAGG-NiV-F、pCAGG-NiV-G和pCAGG-NiV-F pCAGG-NiV-G DNA分别按100μg/只的剂量肌肉注射免疫6周龄BALB/c小鼠,间隔4周加强免疫,第二次加强免疫3周后采血,分离血清备用.分别以重组杆状病毒感染Sf9细胞表达的重组NiV融合蛋白(rNF)和受体结合蛋白(rNG)为包被抗原,应用间接ELISA检测上述质粒DNA免疫血清中的特异性抗体,具有较高的敏感性和特异性.另外,中和试验结果表明,DNA免疫小鼠产生的特异抗体可有效中和NiV囊膜蛋白F和G介导的伪型VSV重组病毒侵入NiV易感宿主细胞的感染性,并且受体结合蛋白G基因DNA诱导中和抗体的滴度高于融合蛋白F基因DNA.结果表明,DNA疫苗具有防制尼帕病毒性脑炎的潜力.

Study on the DNA Immunogenicity of Fusion and Attachment Glycoproteins of Nipah Virus

The two mammalian codon optimized genes, F and G genes of Nipah virus, were generated by assembly PCR, and inserted into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-NiV-F and pCAGG-NiV-G. Syncytium formation was induced in BHK cells by plasmid pCAGG-NiV-F and pCAGG-NiV-G transfection, which indicate recombination proteins F and G were expressed in BHK cell and possessed good biologic activity. Six-week-old female BALB/c mice were intramuscularly primed with 100 microg pCAGG-NiV-F, pCAGG-NiV-G or pCAGG-NiV-F+ pCAGG-NiV-G respectively, and boosted with same dose after 4 weeks. The sera were collected at 3 weeks post second boost. The serum IgG against Nipah virus F and G proteins was detected by indirect ELISA using recombinant Baculovirus expressed Nipah F and G glycoproteins. The results showed that specific antibodies possessed good sensitivity and specificity. Furthermore, the G and F proteins' specific antibodies could neutralize the infectivity of VSVdeltaG* F/G (the NiV F and G envelope glycoproteins psudotyped recombinant vesicular stomatitis virus expressing green fluorescence protein). And, pCAGG-NiV-G also induced higher titer of neutralizing antibody response than pCAGG-NiV-F did. The result indicates that DAN immunization is an efficient vaccine strategy against Nipah virus.