Characterization of Neospora caninum surface protein NcSRS2 based on baculovirus expression system and its application for serodiagnosis of Neospora infection
Characterization of Neospora caninum surface protein NcSRS2 based on baculovirus expression system and its application for serodiagnosis of Neospora infection
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DOI：
10.1128/jcm.39.11.3987-3991.2001
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发表时间：
2001-11-01
影响因子：
9.4
通讯作者：
Nagasawa, H
中科院分区：
文献类型：
--
作者：
Nishikawa, Y;Kousaka, Y;Nagasawa, H
杆状病毒表达系统已被证明是生产重组蛋白的有用工具。本研究鉴定了两种重组病毒产生的犬新孢子虫表面蛋白NcSRS2，并建立了一种酶联免疫吸附试验（ELISA），用于犬新孢子虫感染的血清学诊断。Western blot分析显示，在感染了重组杆状病毒的昆虫细胞中可以检测到两个主要的蛋白带，并且在感染了缺乏疏水c端尾的重组病毒的细胞的培养上清中检测到一个分子量较低的蛋白。n端氨基酸分析表明，分泌的NcSRS2缺少6 kDa的n端信号肽。此外，利用表达全长NcSRS2基因的重组杆状病毒感染的昆虫细胞的清洁可溶性蛋白，建立了一种基于特异性和反应性的ELISA系统，该系统对刚地弓形虫、海氏Hammondia heydorni和犬齿弓形虫的抗血清具有特异性。Anti-N。小鼠、狗和牛血清在Western blots上识别重组NcSRS2。此外，我们已经证明，开发的ELISA系统一致地区分间接荧光抗体测试（IFAT）阳性的牛血清与IFAT阴性的血清。上述结果表明，采用杆状病毒表达的NcSRS2酶联免疫吸附试验可有效、可靠地诊断犬链球菌感染。
The baculovirus expression system has proved to be a useful tool for the production of recombinant proteins. Here we have characterized the Neospora caninum surface protein NcSRS2 produced by two types of the recombinant virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using recombinant NcSRS2 for the serologic diagnosis of Neospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each recombinant baculovirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the recombinant virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the recombinant baculovirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera against Toxoplasma gondii, Hammondia heydorni, or N. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculovirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis of N. caninum infection.