尼帕病毒融合蛋白和受体结合蛋白基因DNA免疫的研究  

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

作　　者：王喜军[1] 葛金英[1] 王清华[2] 胡森[1] 林祥梅[3] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001 [2]农业部青岛动物检疫所国家外来病诊断中心,青岛266032 [3]中国检验检疫科学研究院,北京100025

出　　处：《病毒学报》2008年第1期47-52,共6页Chinese Journal of Virology

基　　金：国家“攻关”项目(2004BA519A19,2005BA711A10);“973”项目(2005CB523200);国家科技支撑计划项目(2006BAD06A00)

摘　　要：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-GDNA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。The two mammalian codon optimized genes, F and G genes of Nipah virus, were generated by as sembly PCR, and inserted into mammalian expression vector pCAGGS under chicken β-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 intra muscularly primed with 100μg pCAGG-NiV-F, pCAGG-NiV-G or pCAGG-NiV F＋ pCAGG-NiV-G re spectively, 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 VSVΔG^＊ 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.

关 键 词：尼帕病毒DNA免疫 F蛋白 G蛋白 

分 类 号：Q786[生物学—分子生物学]