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首页> 外文期刊>Infection and immunity >Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b.
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Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b.

机译：乙型流感嗜血杆菌热可修饰主要外膜蛋白编码基因的克隆和表达。

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摘要

One major outer membrane protein (P1) of Haemophilus influenzae type b (Hib), with an apparent molecular weight of 34,000 (34K) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), has been shown to be heat modifiable. After heating at 100 degrees C for 5 min in 2% SDS, the P1 protein exhibits an apparent molecular weight of 49,000 (49K) in SDS-PAGE. Monoclonal antibodies (MAbs) reactive with P1 bound to the surface of Hib, and one of these MAbs had a protective effect against the development of Hib bacteremia in an animal model for invasive Hib disease. A 6-kilobase Hib DNA insert containing the gene encoding this P1 protein was cloned into Escherichia coli by using the gamma gt11 expression vector. Recombinant phage expressing P1 were identified by screening phage plaques with a MAb directed against the P1 protein. Expression of the P1 protein by an E. coli lysogen carrying the recombinant phage was independent of both vegetative phage growth and induction of lacZ gene-directed transcription of the Hib DNA insert. The Hib DNA insert encoding the P1 protein was subcloned into the plasmid vector pBR322, and a transformant containing the recombinant plasmid pFRG100 was identified with the P1 protein-directed MAb in a colony blot-radioimmunoassay. Western blot (immunoblot) analysis determined that the recombinant P1 protein possessed heat-modifiability characteristics identical to those of the native Hib protein. The P1 protein was expressed on the surface of both the E. coli lysogen containing the recombinant phage and the E. coli transformant containing pFRG100. Western blot analysis of acute- and convalescent-phase sera from infants with Hib meningitis showed that antibodies in the convalescent-phase sera recognized the P1 protein expressed by the E. coli transformant containing pFRG100. The availability of this cloned Hib DNA insert encoding the Hib P1 protein and the expression of this protein on the surface of recombinant E. coli should facilitate the investigation of P1 for both its vaccinogenic potential and its functional role in the outer membrane of Hib.
机译：通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，表观分子量为34,000（34K）的一种主要的b型流感嗜血杆菌外膜蛋白（P1）具有热修饰性。 。在2％SDS中于100摄氏度加热5分钟后，P1蛋白在SDS-PAGE中的表观分子量为49,000（49K）。与P1反应的单克隆抗体（MAb）与Hib的表面结合，其中一种MAb在侵袭性Hib疾病动物模型中具有对抗Hib菌血症发展的保护作用。通过使用γgt11表达载体，将包含编码该P1蛋白的基因的6-千碱基Hib DNA插入物克隆到大肠杆菌中。通过用针对P1蛋白的MAb筛选噬菌斑来鉴定表达重组P1的噬菌体。携带重组噬菌体的大肠杆菌溶原菌对P1蛋白的表达既独立于营养噬菌体的生长，又不受Hib DNA插入物lacZ基因定向转录的诱导。将编码P1蛋白的Hib DNA插入片段亚克隆到质粒载体pBR322中，并在菌落印迹放射免疫分析中用P1蛋白定向的MAb鉴定出含有重组质粒pFRG100的转化体。 Western印迹（免疫印迹）分析确定重组P1蛋白具有与天然Hib蛋白相同的热修饰特性。 P1蛋白在含有重组噬菌体的大肠杆菌溶原菌和含有pFRG100的大肠杆菌转化体的表面表达。对患有Hib脑膜炎的婴儿进行的急性期和恢复期血清的蛋白质印迹分析表明，恢复期血清中的抗体识别包含pFRG100的大肠杆菌转化子表达的P1蛋白。编码Hib P1蛋白的这种克隆Hib DNA插入片段的可用性以及该蛋白在重组大肠杆菌表面上的表达应有助于研究P1的致癌潜力及其在Hib外膜中的功能。

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