Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay
Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay
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DOI：
10.1128/jcm.39.7.2603-2609.2001
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发表时间：
2001-07-01
影响因子：
9.4
通讯作者：
Mikami, T
中科院分区：
文献类型：
--
作者：
Fukumoto, S;Xuan, X;Mikami, T
用B法筛选吉氏巴氏巴氏杆菌裂殖子cDNA表达文库。吉布森感染的狗血清克隆了编码50-kDa蛋白的cDNA并命名为P50基因。P50基因全长1,922 bp。计算机分析表明，P50基因的序列包含一个1,401 bp的开放阅读框架，编码能力约为50 kDa。P50基因的完整基因组核苷酸序列已被分析，并显示含有一个37 bp的单个内含子。Southern杂交分析表明P50基因在B中以单拷贝存在。Gibsoni基因组B的天然P50蛋白。gibsoni，分子量为50 M）a，用抗重组P50小鼠血清通过Western印迹法鉴定。激光共聚焦显微镜分析显示P50蛋白定位于B细胞表面。吉布森裂殖子用杆状病毒在昆虫细胞中表达的重组P50蛋白作为抗原进行酶联免疫吸附试验（ELISA）。ELISA能区分B型。gibsoni感染的狗血清和B.犬感染的犬血清或未感染的犬血清。此外，在实验感染B的狗中，针对重组P50蛋白的抗体应答一直维持到感染的慢性阶段。Gibsoni开发的。这些结果表明，重组P50蛋白可能是一个有用的诊断试剂，用于检测抗体的B。狗的吉布森
A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in the B. gibsoni genome. The native P50 protein of B. gibsoni with a molecular mass of 50 M)a was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface of B. gibsoni merozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected with B. gibsoni was developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies to B. gibsoni in dogs.