McCarvil J, McKenna A J, Grief C, Hoy C S, Sesardic D, Maiden M C, Feavers I M
Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Hertfordshire, UK.
Mol Microbiol. 1993 Oct;10(1):203-13. doi: 10.1111/j.1365-2958.1993.tb00916.x.
The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS-PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.
1类外膜蛋白（OMP）是脑膜炎奈瑟菌的主要可变表面抗原，是目前正在进行现场试验的新型脑膜炎球菌疫苗的一个组成部分。该蛋白的血清学变体也用于脑膜炎球菌的血清亚型分类。导致该蛋白抗原变体的大多数氨基酸变化发生在两个可变区（VR1和VR2），这两个可变区被认为在细胞表面形成环。聚合酶链反应（PCR）用于从脑膜炎奈瑟菌菌株M1080的染色体DNA中扩增编码VR1和VR2的核苷酸序列。将这些序列读框克隆到大肠杆菌表达载体pAJC264的lamB基因中。使用单克隆抗体的全细胞酶联免疫吸附测定（ELISA）和SDS-PAGE证实，诱导后，携带这些构建体的大肠杆菌菌株表达含有脑膜炎奈瑟菌表面环的杂交LamB蛋白。这些菌株用于免疫兔子，所得的多克隆抗血清与参考菌株M1080（P1.7）的1类OMP特异性反应。用这些抗血清对脑膜炎球菌细胞进行免疫金标记和全细胞斑点印迹分析表明，可变表位暴露在细胞表面，并证实该方法可用于获得血清亚型特异性抗血清。通过抗血清与重叠合成肽的反应确定其结合谱，并将其反应性与相关血清亚型特异性单克隆抗体的反应性进行比较。该方法成功地用于产生针对另外两种1类OMP VR2的抗血清。针对包括P1.1表位的VR2产生的第四种抗血清不是亚型特异性的。
