Monoclonal antibody selection and analysis of a recombinant DNA-derived surface immunogen of Treponema pallidum expressed in Escherichia coli.
Monoclonal antibody selection and analysis of a recombinant DNA-derived surface immunogen of Treponema pallidum expressed in Escherichia coli.
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大肠杆菌中表达的梅毒螺旋体重组 DNA 衍生表面免疫原的单克隆抗体选择和分析。
DOI：
10.1128/iai.52.1.110-119.1986
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发表时间：
1986
影响因子：
3.1
通讯作者：
Norgard,MV
中科院分区：
文献类型：
--
作者：
Swancutt,MA;Twehous,DA;Norgard,MV
针对梅毒螺旋体34千道尔顿（kDa）表面免疫原的单克隆抗体用于选择12个独特的T。表达重组形式的34-kDa免疫原的含有苍白球DNA的大肠杆菌重组克隆。克隆的表型取决于宿主细胞中重组质粒的存在。质粒DNA的限制性内切酶分析和Southern杂交表明，所有重组克隆都含有T.苍白球起源进一步的杂交分析表明，克隆的T.苍白球DNA序列是T.苍白球基因组DNA排列。从混合免疫兔血清中纯化的免疫球蛋白G（IgG）与克隆反应，而从混合正常兔血清中纯化的IgG不与克隆反应。免疫学实验和Southern杂交结果表明，T.苍白球亚种pertenue，但它是不存在的四种非致病性密螺旋体测试，以及从正常兔睾丸组织匀浆。E.用[35 S]蛋氨酸标记的大肠杆菌克隆，然后用单克隆抗体进行放射免疫沉淀，结果显示35 S标记的重组体和125 I标记的天然（T.苍白球）形式的抗原具有相同的电泳迁移率。E.大肠杆菌中外源基因的表达与外源基因相对于载体DNA的方向无关。T.在放线菌酮存在下，用[35 S]甲硫氨酸代谢标记后，苍白球也产生了明显相同的免疫沉淀34-kDa抗原。34-kDa免疫原对致病性密螺旋体的表观特异性及其在T。苍白球证明了对该抗原及其相应基因的更深入研究。
Monoclonal antibodies directed against a 34-kilodalton (kDa) surface immunogen of Treponema pallidum were used to select 12 unique T. pallidum DNA-containing Escherichia coli recombinant clones expressing the recombinant form of the 34-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmids in the host cell. Restriction enzyme analyses and Southern hybridization of plasmid DNA demonstrated that all recombinant clones contained common DNA sequences of T. pallidum origin. Further hybridization analyses revealed that the cloned T. pallidum DNA sequences were an accurate representation of the T. pallidum genomic DNA arrangement. Purified immunoglobulin G (IgG) from pooled immune rabbit serum reacted with the clones, while IgG from pooled normal rabbit serum did not. Results of immunological experiments and Southern hybridization indicated that a similar 34-kDa immunogen was present in T. pallidum subsp. pertenue, but it was absent from four species of nonpathogenic treponemes tested, as well as from homogenates of normal rabbit testicular tissue. Metabolic labeling of the E. coli clones with [35S]methionine followed by radioimmunoprecipitation with monoclonal antibodies revealed that the 35S-labeled recombinant and 125I-labeled native (T. pallidum) forms of the antigen had identical electrophoretic mobilities. The production of a complete antigen by E. coli was independent of the orientation of the foreign gene sequence with respect to vector DNA. T. pallidum also produced an apparently identical immunoprecipitable 34-kDa antigen after metabolic labeling with [35S]methionine in the presence of cycloheximide. The apparent specificity of the 34-kDa immunogen for pathogenic treponemes and its native cell surface association on T. pallidum justifies a more intense study of this antigen and its corresponding gene.