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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis.
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Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis.

机译：两个重组新孢子虫蛋白片段的克隆，鉴定及其在牛新孢子虫病血清学诊断中的应用。

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Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora lambda gt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecular masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined "gold standard" panel of bovine sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate-based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.
机译：牛新孢子虫病会导致牛的胎儿流产和/或先天性神经系统疾病。对于这种寄生虫疾病的血清诊断，从牛新孢子虫gt11文库中鉴定了两个免疫优势克隆，并进行了表征，并表达为重组蛋白，用于开发酶联免疫吸附测定（ELISA）。当从pRSET表达载体表达为组氨酸融合蛋白时，这两个克隆分别命名为N54和N57，分别为29和20 kDa。重组蛋白N54的抗体从分子质量为97、87、77、67和64 kDa的新孢子虫速殖子裂解物中识别出五个主要谱带。重组蛋白N57的抗体可识别四个主要条带，分子量分别为34、31、30和28 kDa。当通过免疫印迹对来自确诊的新孢子虫阳性和新孢子虫阴性牛的牛血清的确定的“金标准”组进行鉴定时，在60个新孢子虫阳性血清样品中有57个识别的蛋白质具有N54七联蛋白的分子量。绑定到N57四联体更具可变性。使用相同的金标准板评估和比较基于N54的ELISA，基于N57的ELISA和基于全速殖子裂解物的ELISA。敏感性和特异性分别为95％和96％（N54 ELISA），82％和93％（N57 ELISA）以及74％和93％（裂解物ELISA）。因此，与基于全速殖子裂解物的ELISA相比，两种基于重组蛋白的ELISA均具有更高的灵敏度和更高或相同的特异性，可用于代替全速殖子裂解物ELISA进行牛新孢子虫病的血清学诊断。

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