van Embden J D, van der Donk H J, van Eijk R V, van der Heide H G, de Jong J A, van Olderen M F, Osterhaus A B, Schouls L M
Infect Immun. 1983 Oct;42(1):187-96. doi: 10.1128/iai.42.1.187-196.1983.
A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.
通过将经SauI酶切的梅毒螺旋体DNA克隆到黏粒载体pHC79中，构建了大肠杆菌K-12中的梅毒螺旋体DNA基因文库。在研究的800个克隆中，有16个产生了一种或多种能与梅毒患者抗体发生反应的抗原。根据十二烷基硫酸钠-聚丙烯酰胺凝胶上产生的抗原的分离模式，在这16个克隆中区分出了6种不同的表型。这些抗原也与抗梅毒螺旋体兔血清发生反应。在正常兔血清和非梅毒患者血清中未发现针对克隆抗原的抗体。大肠杆菌K-12重组DNA克隆产生的抗原在十二烷基硫酸钠-聚丙烯酰胺凝胶中与从梅毒螺旋体细菌中提取的抗原迁移位置相同，这表明梅毒螺旋体DNA在大肠杆菌K-12中得到了良好表达。几个黏粒重组质粒已被亚克隆，得到了更小的梅毒螺旋体重组质粒，这些质粒在细胞中更稳定地维持，并产生更多的梅毒螺旋体抗原。制备了针对梅毒螺旋体的单克隆抗体，其中一个杂交瘤产生的抗体不仅能与梅毒螺旋体的一种抗原发生反应，还能与大肠杆菌克隆之一产生的抗原发生反应。
