Ala' Aldeen D A, Westphal A H, De Kok A, Weston V, Atta M S, Baldwin T J, Bartley J, Borriello S P
Department of Microbiology, University Hospital, Queen's Medical Centre, Nottingham.
J Med Microbiol. 1996 Dec;45(6):419-32. doi: 10.1099/00222615-45-6-419.
A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity of this domain. Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infected with homologous or heterologous meningococci or other neisserial species. These results have important implications for the understanding of PDHC and the design of future outer membrane vesicle-based vaccines.
用兔多克隆抗血清（R - 70）筛选脑膜炎奈瑟菌的lambdaZap - II表达文库，该抗血清是针对通过从制备性SDS - 聚丙烯酰胺凝胶上洗脱而从外膜囊泡中纯化的约70 kDa蛋白质产生的。分离出选定的克隆，进一步纯化，并切除其重组pBluescript SKII质粒。对阳性克隆的克隆DNA插入片段进行测序和分析。鉴定出四个开放阅读框（ORF），其中三个与许多原核和真核物种的丙酮酸脱氢酶复合物（PDHC）的丙酮酸脱氢酶（E1p）、二氢硫辛酰胺乙酰转移酶（E2p）和二氢硫辛酰胺脱氢酶（E3）组分具有高度同源性。序列分析表明，脑膜炎奈瑟菌E2p（Men - E2p）包含两个N端硫辛酰结构域、一个E1/E3结合结构域和一个催化结构域。这些结构域由富含丙氨酸、脯氨酸和带电荷残基的铰链区隔开。在E3组分的N端发现了另一个与Men - E2p硫辛酰结构域具有高度序列相似性的硫辛酰结构域。在编码E2p和E3组分的ORF之间发现了另一个编码16.5 kDa蛋白质的ORF。通过免疫和生化分析证实了表达和纯化的异源Men - E2p作为二氢硫辛酰胺乙酰转移酶的身份和功能特性。N端氨基酸分析证实了DNA衍生的成熟蛋白的序列。纯化的Men - E2p与针对整个E2p分子以及针对棕色固氮菌E2p的硫辛酰结构域产生的单特异性抗血清发生反应。相反，针对Men - E2p产生的兔抗血清与棕色固氮菌、大肠杆菌和淋病奈瑟菌的蛋白提取物以及通过有限蛋白酶解获得的E2p的硫辛酰和催化结构域发生反应。相比之下，原始的R - 70抗血清几乎只与硫辛酰结构域发生反应，表明该结构域具有很强的免疫原性。在感染同源或异源脑膜炎奈瑟菌或其他奈瑟菌属物种的患者和动物（兔子和小鼠）中检测到了针对Men - E2p的抗体。这些结果对理解PDHC以及未来基于外膜囊泡的疫苗设计具有重要意义。
