High-level expression of truncated surface antigen P50 of Babesia gibsoni in insect cells by baculovirus and evaluation of its immunogenicity and antigenicity
High-level expression of truncated surface antigen P50 of Babesia gibsoni in insect cells by baculovirus and evaluation of its immunogenicity and antigenicity
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DOI：
10.1128/cdli.10.4.596-601.2003
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发表时间：
2003-07-01
期刊：
影响因子：
--
通讯作者：
Suzuki, H
中科院分区：
文献类型：
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作者：
Fukumoto, S;Xuan, XN;Suzuki, H
此前，我们鉴定了吉布氏巴贝虫的免疫显性抗原 P50。在本研究中，编码无C端疏水区（29个氨基酸[aa]）的截短P50（rP50t）的基因通过重组杆状病毒在昆虫细胞中表达。高度疏水的C端20-aa区域似乎是一个跨膜区域，rP50t有效分泌到感染重组杆状病毒的昆虫细胞上清液中的事实证明了这一点。 rP50t的N端氨基酸序列分析表明N端19个氨基酸充当信号肽。每108个重组杆状病毒感染的细胞中rP50t的表达水平高达2mg。通过小鼠免疫试验评估rP50t的免疫原性。用 rP50t 免疫的小鼠诱导了针对吉布氏芽孢杆菌裂殖子的高水平抗体滴度。在小鼠中产生 rP50t 的单克隆抗体 (MAb)，以确定 P50 的免疫原性区域。所有五种单克隆抗体识别的表位均位于氨基酸 190 和 273 之间，表明 P50 的中心部分是一个高度免疫原性的区域。使用酶联免疫吸附测定 (ELISA) 评估 rP50t 的诊断潜力。 ELISA 能够清楚地区分 (P < 0.0001) 吉布氏芽胞杆菌感染的狗血清和犬芽孢杆菌感染的狗血清或未感染的狗血清。我们的结果表明，rP50t 可能提供一种有用的潜在免疫原性试剂，用于诊断和作为亚单位疫苗来控制狗的吉布氏伯克体感染。
Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 108 cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.