Wang Xi-Jun, Ge Jin-Ying, Wang Qing-Hua, Hu Sen, Lin Xiang-Mei, Bu Zhi-Gao
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Bing Du Xue Bao. 2008 Jan;24(1):47-52.
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.
通过组装PCR生成了尼帕病毒的两个哺乳动物密码子优化基因F和G基因，并将其插入鸡β-肌动蛋白启动子控制下的哺乳动物表达载体pCAGGS中，构建了pCAGG-NiV-F和pCAGG-NiV-G。通过转染质粒pCAGG-NiV-F和pCAGG-NiV-G在BHK细胞中诱导形成多核巨细胞，这表明重组蛋白F和G在BHK细胞中表达并具有良好的生物学活性。分别用100μg pCAGG-NiV-F、pCAGG-NiV-G或pCAGG-NiV-F + pCAGG-NiV-G对6周龄雌性BALB/c小鼠进行肌肉初次免疫，并在4周后用相同剂量进行加强免疫。在第二次加强免疫后3周收集血清。使用重组杆状病毒表达的尼帕F和G糖蛋白，通过间接ELISA检测针对尼帕病毒F和G蛋白的血清IgG。结果表明特异性抗体具有良好的敏感性和特异性。此外，G和F蛋白的特异性抗体能够中和VSVdeltaG* F/G（表达绿色荧光蛋白的尼帕病毒F和G包膜糖蛋白假型重组水疱性口炎病毒）的感染性。并且，pCAGG-NiV-G诱导的中和抗体反应滴度也高于pCAGG-NiV-F。结果表明DNA免疫是一种针对尼帕病毒的有效疫苗策略。
