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首页> 外文期刊>Infection and immunity >Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae.
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Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae.

机译：保留对从不可分型流感嗜血杆菌外膜制备聚丙烯酰胺凝胶电泳分离的蛋白质的免疫应答。

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Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell-mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.
机译：已通过制备型聚丙烯酰胺凝胶电泳（PAGE）从不可分型流感嗜血杆菌菌株中纯化了分子量分别为26和28 kDa的外膜蛋白P2，P4和P6以及两种。通过天然PAGE和SDS-PAGE分别从三株不可分型的流感嗜血杆菌和一株b H型菌株中纯化了分子量为16 kDa（由十二烷基硫酸钠[SDS] -PAGE确定）的外膜蛋白P6。流感。从每个菌株纯化需要相同的条件。通过研究用不完全弗氏佐剂中的P6免疫到Peyer斑块中的P6免疫的大鼠的免疫反应，评估了通过这些方法分离的蛋白质的适用性。通过天然PAGE或SDS-PAGE纯化的P6与通过凝胶过滤和阴离子交换色谱纯化的P6在增强活细菌的肺部清除能力上没有显着差异。这项研究还研究了SDS对体内P2免疫反应的影响，以及两性试剂Zwittergent和月桂基肌氨酸钠对体外外膜蛋白淋巴细胞增殖反应的影响。发现免疫乳剂中SDS的存在增强了抗原特异性细胞介导的应答，但是抑制了抗原特异性抗体应答。外膜蛋白制剂中残留的两性离子残留痕迹抑制了抗原特异性细胞介导的增殖，而用月桂基肌氨酸钠提取外膜蛋白并没有抑制抗原特异性增殖。这些结果表明，制备性PAGE是从流感嗜血杆菌外膜中纯化蛋白质的合适方法，这是研究其作为疫苗候选物的免疫学意义所必需的，并且蛋白质纯化过程中使用的试剂痕迹可能对确定流感疫苗的发生起重要作用。体内和体外研究的成功。

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