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首页> 外文期刊>Infection and immunity >Molecular cloning and analysis of recombinant major antigens of Ehrlichia risticii.
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Molecular cloning and analysis of recombinant major antigens of Ehrlichia risticii.

机译：重组埃里希氏菌的主要抗原的分子克隆和分析。

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The genome of Ehrlichia risticii, the etiologic agent of Potomac horse fever, was cloned in the lambda gt11 expression vector. The efficiency of recombinant phage production with different restriction fragments of E. risticii DNA was generally between 20 and 95%. The antigen-positive frequency, detected by immunoscreening with E. risticii antibodies, was between 8 and 40 per 10(4) recombinants. Four (70, 55, 51, and 44 kDa) major antigens of E. risticii were identified from the recombinant phages by using recombinant antigen-selected monospecific antibodies. Characterization of three (70, 55, and 44 kDa) of these recombinant antigens indicated that the 70- and 44-kDa polypeptides were beta-galactosidase fusion products that were dependent on isopropylthiogalactoside induction for expression; they contained about 50 and 73%, respectively, of the native polypeptides. The 55-kDa antigen was a nonfusion protein expressed independently of isopropylthiogalactoside induction; it was a complete protein with a molecular weight identical to that of its native counterpart. The cloned E. risticii DNAs from of the recombinants expressing 70-, 55-, and 44-kDa proteins were 3.5, 3.9, and 4.8 kb, respectively, in size, and they were unique. The insert DNAs hybridized to multiple restriction fragments of the genomic DNA, the sum of the sizes of which was much greater than that of the corresponding insert. Mice immunized with the affinity-purified 55-kDa recombinant antigen produced a high titer of antibody in serum as measured by an enzyme-linked immunosorbent assay and gave a monospecific reaction by Western immunoblotting. Challenge infection of these immunized mice showed low protection from clinical infection.
机译：将波托马克马热的病原体埃里希氏菌的基因组克隆到λgt11表达载体中。具有不同的埃希氏大肠杆菌DNA限制片段的重组噬菌体生产效率通常在20％至95％之间。通过用埃希氏大肠杆菌抗体进行免疫筛选检测到的抗原阳性频率为每10（4）个重组体8到40个之间。通过使用重组抗原选择的单特异性抗体，从重组噬菌体中鉴定出大肠杆菌的四个主要抗原（70、55、51和44 kDa）。这些重组抗原中的三个（70、55和44 kDa）的特征表明70和44 kDa多肽是依赖于异丙基硫代半乳糖苷诱导表达的β-半乳糖苷酶融合产物。它们分别包含约50％和73％的天然多肽。 55kDa抗原是一种非融合蛋白，独立于异丙基硫代半乳糖苷的诱导表达。它是一种完整的蛋白质，分子量与其天然对应物的分子量相同。从表达70-，55-和44-kDa蛋白的重组体中克隆的埃希氏大肠杆菌DNA的大小分别为3.5、3.9和4.8kb，并且它们是独特的。插入物DNA与基因组DNA的多个限制性片段杂交，其大小之和远大于相应插入物的大小之和。用亲和纯化的55-kDa重组抗原免疫的小鼠在血清中可产生高滴度的抗体（如通过酶联免疫吸附测定法测定），并通过Western免疫印迹进行单特异性反应。这些免疫小鼠的激发感染显示出对临床感染的低保护。

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