Muldrow L L, Ibeanu G C, Lee N I, Bose N K, Johnson J
FEBS Lett. 1987 Mar 23;213(2):249-53. doi: 10.1016/0014-5793(87)81500-4.
Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.
艰难梭菌的毒素A已被纯化并制备出单特异性抗血清。开发了一种可靠的分离和限制艰难梭菌染色体DNA的程序，该程序允许构建λgt11基因组文库。使用抗毒素A筛选了约35,000个噬菌斑，结果鉴定出一个稳定的阳性克隆λcd19。通过亲和纯化针对λcd19基因产物的毒素A抗体，并使用这些选定的抗体探测纯化毒素A的蛋白质免疫印迹，来确定λcd19中分离的毒素A基因片段的免疫学同一性。通过限制性消化以及该克隆与艰难梭菌染色体消化物的杂交，证明λcd19中的插入片段是一个0.3 kb的片段。λcd19中毒素A基因片段编码的肽对3T3哺乳动物组织培养细胞没有细胞毒性。
