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首页> 外文期刊>Infection and immunity >Cloning and expression of the CAMP factor of group B streptococci in Escherichia coli.
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Cloning and expression of the CAMP factor of group B streptococci in Escherichia coli.

机译：B组链球菌CAMP因子的克隆及在大肠杆菌中的表达。

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The genetic determinant of the CAMP factor from a strain of group B streptococcus (GBS; Streptococcus agalactiae) was cloned in Escherichia coli. Total cell DNA from the GBS strain R268 was used to construct a gene bank with bacteriophage lambda EMBL4 in the E. coli K-12 strain LE392. Recombinant phage plaques were detected by immunoblots by using a specific antiserum raised against purified CAMP factor. Two hybrid phages showing expression of CAMP factor were identified. Subcloning the CAMP gene (cfb) into the high-copy-number vector pUC8 resulted in highly unstable plasmids. Therefore, subcloning was performed with the low-copy-number vector pLG339 resulting in the stable recombinant plasmids pCO61 and pCO62 which lead to expression of CAMP protein first identified by colony immunoblotting. Western blot (immunoblot) analysis revealed a similar CAMP protein pattern in lambda EMBL4 recombinant phage lysates (molecular weight, 22,000 to 24,000) as compared to that obtained from a GBS culture supernatant (molecular weight, 22,000 to 26,000) but a different CAMP protein pattern (molecular weight, 20,000 to 23,000) from lysates of E. coli carrying pCO61 or pCO62. To study the excretion of the CAMP protein we performed a semi-quantitative dot blot analysis of proteins recovered from cell fractions and supernatants of the E. coli recombinant clones. In contrast to GBS R268, where the CAMP factor is readily excreted, the CAMP protein is not excreted in E. coli clones containing pCO61 and pCO62 but is found associated with the cell fractions.
机译：来自B组链球菌（GBS;无乳链球菌）菌株的CAMP因子的遗传决定子被克隆到大肠杆菌中。来自GBS菌株R268的总细胞DNA用于构建大肠杆菌K-12菌株LE392中带有噬菌体λEMBL4的基因库。通过使用针对纯化的CAMP因子的特异性抗血清，通过免疫印迹检测重组噬菌体噬菌斑。鉴定出两个显示CAMP因子表达的杂合噬菌体。将CAMP基因（cfb）亚克隆到高拷贝数载体pUC8中导致了高度不稳定的质粒。因此，用低拷贝数的载体pLG339进行亚克隆，得到稳定的重组质粒pCO61和pCO62，其导致首先通过菌落免疫印迹鉴定的CAMP蛋白的表达。 Western blot（免疫印迹）分析显示，与从GBS培养上清液（分子量，22,000至26,000）相比，λEMBL4重组噬菌体裂解物（分子量，22,000至24,000）中的CAMP蛋白模式相似。 （分子量为20,000至23,000）来自带有pCO61或pCO62的大肠杆菌裂解物。为了研究CAMP蛋白的排泄，我们对从大肠杆菌重组克隆的细胞级分和上清液中回收的蛋白进行了半定量斑点印迹分析。与GBS R268相比，CAMP因子易于排出，而CAMP蛋白在含有pCO61和pCO62的大肠杆菌克隆中不排出，但与细胞组分有关。

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