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首页> 外文期刊>Infection and immunity >Cloning and expression in Escherichia coli of the Ibc protein genes of group B streptococci: binding of human immunoglobulin A to the beta antigen.
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Cloning and expression in Escherichia coli of the Ibc protein genes of group B streptococci: binding of human immunoglobulin A to the beta antigen.

机译：B链球菌的Ibc蛋白基因的克隆和在大肠杆菌中的表达：人免疫球蛋白A与β抗原的结合。

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摘要

Total-cell DNA isolated from a highly virulent serotype Ic strain of a group B streptococcus was used to construct a gene bank with bacteriophage lambda L47.1 in Escherichia coli K-12. Recombinant phage plaques in the bank were immunoblotted by using anti-alpha- and anti-beta-specific antibodies directed towards the Ibc proteins purified from the streptococcal cell surface, and hybrid phages expressing the alpha protein (lambda alpha +) and the beta protein (lambda beta +) were identified. DNA inserts in these phages were subcloned into E. coli high-copy-number plasmid vectors to produce stable alpha + (pPHC10) and beta + (pPHC8 and pPHC33) recombinant plasmids, and restriction maps of the cloned streptococcal sequences were constructed. Antibodies against the two Streptococcus-derived proteins reacted with high-molecular-weight polypeptides made in E. coli cells carrying the corresponding hybrid plasmids and with several degradation peptides from them. A 190-kilodalton alpha protein, previously undetected, was identified; this species may be the native alpha protein or a precursor of it. In addition, mutagenesis of the cloned sequences was carried out by using the omega fragment to determine the direction of transcription. In E. coli, the beta protein, but not the alpha protein, bound human immunoglobulin A (IgA) in Western blots, and neither protein bound IgG or IgM.
机译：从B组链球菌的强毒血清型Ic株中分离出的总细胞DNA被用于构建大肠杆菌K-12中带有噬菌体λL47.1的基因库。通过使用针对从链球菌细胞表面纯化的Ibc蛋白的抗α和抗β特异性抗体以及表达α蛋白（λalpha +）和β蛋白（）的杂合噬菌体，对库中的重组噬菌体斑进行免疫印迹。确定了lambda beta +）。将这些噬菌体中的DNA插入片段亚克隆到大肠杆菌高拷贝数质粒载体中，以产生稳定的α+（pPHC10）和β+（pPHC8和pPHC33）重组质粒，并构建了克隆的链球菌序列的限制性酶切图。针对两种链球菌衍生蛋白的抗体与在大肠杆菌细胞中制备的高分子量多肽反应，该多肽携带相应的杂合质粒，并与其中的一些降解肽反应。鉴定出先前未发现的190千达尔顿α蛋白。该物种可能是天然的α蛋白或其前体。另外，通过使用ω片段确定转录方向来诱变克隆的序列。在大肠杆菌中，在蛋白质印迹法中，β蛋白而非α蛋白与人免疫球蛋白A（IgA）结合，而蛋白与IgG或IgM均不结合。

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