Beguin P, Cornet P, Millet J
Biochimie. 1983 Aug-Sep;65(8-9):495-500. doi: 10.1016/s0300-9084(83)80131-x.
The endoglucanase encoded by the celB gene of Clostridium thermocellum was purified from an E. coli strain carrying and expressing the C. thermocellum gene cloned in the plasmid pBR322. The preparation showed two active bands, with Mr 55,000 and 53,000, presumably derived from the primary translation product by proteolysis. Specific antiserum raised against these bands was used to identify the corresponding antigen in the culture supernatant of C. thermocellum: in a double immunodiffusion test (Ouchterlony), a precipitin line was observed which fused completely with that formed by an E. coli extract containing endoglucanase B expressed from the cloned gene. Proteins from C. thermocellum supernatant were further analyzed by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet. After incubating the nitrocellulose blot with antiserum and subsequently with 125I-labeled protein A, a band with Mr 66,000, corresponding to the celB gene product expressed by C. thermocellum, was detected by autoradiography.
从携带并表达克隆于质粒pBR322中的嗜热栖热放线菌基因的大肠杆菌菌株中纯化出嗜热栖热放线菌celB基因编码的内切葡聚糖酶。该制剂显示出两条活性带，分子量分别为55,000和53,000，推测是由初级翻译产物经蛋白水解产生的。针对这些条带产生的特异性抗血清用于鉴定嗜热栖热放线菌培养上清液中的相应抗原：在双向免疫扩散试验（欧氏试验）中，观察到一条沉淀线，它与由含有从克隆基因表达的内切葡聚糖酶B的大肠杆菌提取物形成的沉淀线完全融合。通过SDS-聚丙烯酰胺凝胶电泳对嗜热栖热放线菌上清液中的蛋白质进行进一步分析，并转移到硝酸纤维素膜上。在用抗血清孵育硝酸纤维素印迹，随后用125I标记的蛋白A孵育后，通过放射自显影检测到一条分子量为66,000的条带，对应于嗜热栖热放线菌表达的celB基因产物。
