Norgard M V, Miller J N
Infect Immun. 1983 Nov;42(2):435-45. doi: 10.1128/iai.42.2.435-445.1983.
Hybrid pBR322 plasmid clone banks comprised of more than 125,000 recombinant DNA clones and representing the entire Treponema pallidum Nichols genome were constructed in Escherichia coli K-12 RR1. The two clone banks individually contain over 53,000 and 72,000 recombinant clones. The number average and mass average sizes of the cloned DNA inserts were found to be approximately 12 and 13 kilobase pairs, respectively, indicating the presence of large treponemal DNA inserts in a majority of recombinant clones. To detect E. coli clones synthesizing T. pallidum antigens as hybrid plasmid gene translation products in the clone bank, a simplified, direct, solid-phase radioimmuno-colony blot (RICB) assay was developed employing immunoglobulin G antibody isolated from anti-T. pallidum immune rabbit serum. Clones with positive reactivities in the RICB assay were isolated at frequencies of 0.1 to 0.2%. One isolated RICB-positive clone, designated RICB2-1, produced a very strong signal in the RICB assay and was subsequently found through E. coli cell-free in vitro transcription/translation analysis to encode the synthesis of two gene translation products with apparent molecular weights of 77,000 and 44,000. The 44,000-dalton protein was effectively immunoprecipitated from [35S]methionine-labeled E. coli clone cells by using either immune rabbit serum (preabsorbed with Treponema phagedenis biotype Reiter antigens) or selected human syphilitic serum, whereas the 77,000-dalton protein was never immunoprecipitable by similar methods. Purified plasmid DNA from clone RICB2-1 contained a treponemal DNA insert of 3.70 kilobase pairs, which was of suitable size to code for the 121-dalton (44 + 77) protein. The insert was also flanked on each end by PstI sites and possessed three internal PstI sites with fragment sizes of 2.15, 1.18, 0.20, and 0.17 kilobase pairs. Purified clone RICB2-1 plasmid DNA was capable of transforming recipient E. coli cells to virtually 100% RICB reactivity, thus substantiating the plasmid-encoded characteristic. Further experiments employing various antisera in radioimmunoprecipitation systems utilizing cell-free in vitro synthesized gene translation products from clone RICB2-1 also provided the first evidence that E. coli may be capable of using endogenous T. pallidum DNA promotors for genetic expression. These studies, amplified by the isolation of a potentially significant immunoprecipitable 44,000-dalton recombinant protein antigen, point to the importance of the "cloned antigen gene" approach for the eventual eludication of specific antigens or immunogens operative in the pathogenesis, immunology, and serodiagnosis of T. pallidum infection.
由超过125,000个重组DNA克隆组成、代表梅毒螺旋体Nichols株全基因组的杂交pBR322质粒克隆文库在大肠杆菌K-12 RR1中构建。这两个克隆文库分别包含超过53,000个和72,000个重组克隆。发现克隆的DNA插入片段的数均大小和质均大小分别约为12和13千碱基对，表明大多数重组克隆中存在大的梅毒螺旋体DNA插入片段。为了在克隆文库中检测作为杂交质粒基因翻译产物合成梅毒螺旋体抗原的大肠杆菌克隆，开发了一种简化、直接的固相放射免疫菌落印迹（RICB）测定法，该方法采用从抗梅毒螺旋体免疫兔血清中分离的免疫球蛋白G抗体。在RICB测定中具有阳性反应性的克隆以0.1%至0.2%的频率被分离出来。一个分离出的RICB阳性克隆，命名为RICB2-1，在RICB测定中产生了非常强的信号，随后通过大肠杆菌无细胞体外转录/翻译分析发现它编码合成两种表观分子量分别为77,000和44,000的基因翻译产物。通过使用免疫兔血清（用噬菌密螺旋体生物型Reiter抗原预先吸收）或选定的梅毒患者血清，从[35S]甲硫氨酸标记的大肠杆菌克隆细胞中有效地免疫沉淀出了44,000道尔顿的蛋白质，而77,000道尔顿的蛋白质从未通过类似方法被免疫沉淀。来自克隆RICB2-1的纯化质粒DNA包含一个3.70千碱基对的梅毒螺旋体DNA插入片段，其大小适合编码121道尔顿（44 + 77）的蛋白质。该插入片段在两端也侧翼有PstI位点，并具有三个内部PstI位点，片段大小分别为2.15、1.18、0.20和0.17千碱基对。纯化的克隆RICB2-1质粒DNA能够将受体大肠杆菌细胞转化为几乎100%的RICB反应性，从而证实了质粒编码的特性。在利用来自克隆RICB2-1的无细胞体外合成基因翻译产物的放射免疫沉淀系统中使用各种抗血清进行的进一步实验也首次证明大肠杆菌可能能够利用内源性梅毒螺旋体DNA启动子进行基因表达。这些研究，通过分离出一种潜在重要的可免疫沉淀的44,000道尔顿重组蛋白抗原得到加强，指出了“克隆抗原基因”方法对于最终阐明在梅毒螺旋体感染的发病机制、免疫学和血清学诊断中起作用的特定抗原或免疫原的重要性。
