Hosamani Madhusudan, Shimizu Shinya, Hirota Jiro, Kokuho Takehiro, Kubota Takayuki, Watanabe Satoko, Ohta Masato, Muneta Yoshihiro, Inumaru Shigeki
National Institute of Animal Health, 3–1–5 Kan-non-dai, Tsukuba, Ibaraki 305–0856, Japan.
J Vet Med Sci. 2011 May;73(5):609-13. doi: 10.1292/jvms.10-0213. Epub 2010 Dec 22.
In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.
在本研究中，对1994年从日本枥木县牛群中分离出的蓝舌病病毒（BTV）21型的群特异性抗原VP7进行了测序和表达特征分析。使用逆转录聚合酶链反应（RT-PCR）从病毒双链RNA合成的cDNA中扩增基因。该分离株的核苷酸序列与其他已发表的BTV VP7序列显示出高度相似性。在病毒多角体蛋白启动子的控制下，通过杆状病毒基因表达系统在昆虫细胞中表达了VP7的全长和C末端截短形式。用VP7特异性单克隆抗体（8A3B.6，ATCC）进行免疫沉淀，证实了全长重组VP7的表达。带有或不带有6x His标签表达的重组蛋白在TN5细胞中显示出良好的表达水平，并在间接ELISA中与单克隆抗体反应良好。然而，带有His标签的C末端截短的VP7未能与该单克隆抗体反应，而当它与感染牛血清反应时，抗原性较差。后者抗原性的降低表明，C末端截短可能通过抑制VP7三聚体结构的形成影响8A3B.6表位的构建。
