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Neisseria meningitidis : the Class I outer membrane protein

Neisseria meningitidis : the Class I outer membrane protein
Neisseria meningitidis : the Class I outer membrane protein

Outer membrane proteins (OMPs) of Neisseria meningitidis may be involved in pathogenic mechanisms and are candidates for development of effective vaccines. A molecular approach was adopted to isolate structural genes encoding meningococcal OMPs, in order to characterise these important surface antigens and to obtain information relevant to vaccine design. A local isolate of N.meningitidis (MC50) was selected; outer membranes were isolated and used to immunise animals to obtain polyclonal anti-outer membrane-complex (OMC) sera. Sera were characterised by ELISA and Western blot analysis; a rabbit serum (R139 B3) was shown to react with several major OMPs and could detect low levels of native OMC antigen. Anti-E.coli activity contained in this serum was effectively removed by absorption with relevant E.coli antigens. A random genomic library of meningococcal genomic fragments generated by partial Sau3A digestion of MC50 DNA was constructed in the λL47.1 vector. This library was screened using the R139 B3 serum for expression of meningococcal IMP antigens; no expression was detected. A second library was constructed by ligation of MC50 genomic DNA fragments generated by EcoR1 digestion into the expression vector λgt11, and screened with the R139 B3 serum. Several expressing recombinants were isolated; further investigations showed these to be identical. A representative, (λA1) was selected for further charcterisation. Subsequent experiments indicated that λA1 was expressing a large proportion of the Class 1 OMP as a transcriptional/translational fusion with the vector lacZ gene. The entire structural gene was obtained and the DNA sequence was determined; the hydropathy profile and structural features of the predicted protein primary sequence were typical of a bacterial OMP. Comparison of the predicted protein sequences of meningococcal Class 1 and gonococcal porin proteins revealed considerable structural homology; immunological cross-reactivity was demonstrated by Western blot analysis. Class 1 protein also shows immunological and structural homology with meningococcal Class 2 porin protein. Class 1 protein may function as an additional meningococcal porin. Genes encoding antigenically distinct Class 1 proteins were isolated from two additional strains of N.meningitidis. Comparison of these three sequences showed extensive homology at the DNA and protein level. Sequence diversity was located in two discrete regions, which are therefore likely to contain epitopes responsible for the antigenic (subtype) heterogeneity of these proteins. Class 1 proteins from all three meningococcal strains were expressed at low levels in E.coli. The apparent molecular weight by SDS-PAGE of the recombinant protein was identical to that of the native protein in the meningococcus, indicating that these proteins are processed and possibly translocated to the E.coli outer membrane.

University of Southampton
Barlow, Ann Katherine
bf113311-38b2-464a-8de8-5aa148f0f697
Barlow, Ann Katherine
bf113311-38b2-464a-8de8-5aa148f0f697

Barlow, Ann Katherine (1989) Neisseria meningitidis : the Class I outer membrane protein. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Outer membrane proteins (OMPs) of Neisseria meningitidis may be involved in pathogenic mechanisms and are candidates for development of effective vaccines. A molecular approach was adopted to isolate structural genes encoding meningococcal OMPs, in order to characterise these important surface antigens and to obtain information relevant to vaccine design. A local isolate of N.meningitidis (MC50) was selected; outer membranes were isolated and used to immunise animals to obtain polyclonal anti-outer membrane-complex (OMC) sera. Sera were characterised by ELISA and Western blot analysis; a rabbit serum (R139 B3) was shown to react with several major OMPs and could detect low levels of native OMC antigen. Anti-E.coli activity contained in this serum was effectively removed by absorption with relevant E.coli antigens. A random genomic library of meningococcal genomic fragments generated by partial Sau3A digestion of MC50 DNA was constructed in the λL47.1 vector. This library was screened using the R139 B3 serum for expression of meningococcal IMP antigens; no expression was detected. A second library was constructed by ligation of MC50 genomic DNA fragments generated by EcoR1 digestion into the expression vector λgt11, and screened with the R139 B3 serum. Several expressing recombinants were isolated; further investigations showed these to be identical. A representative, (λA1) was selected for further charcterisation. Subsequent experiments indicated that λA1 was expressing a large proportion of the Class 1 OMP as a transcriptional/translational fusion with the vector lacZ gene. The entire structural gene was obtained and the DNA sequence was determined; the hydropathy profile and structural features of the predicted protein primary sequence were typical of a bacterial OMP. Comparison of the predicted protein sequences of meningococcal Class 1 and gonococcal porin proteins revealed considerable structural homology; immunological cross-reactivity was demonstrated by Western blot analysis. Class 1 protein also shows immunological and structural homology with meningococcal Class 2 porin protein. Class 1 protein may function as an additional meningococcal porin. Genes encoding antigenically distinct Class 1 proteins were isolated from two additional strains of N.meningitidis. Comparison of these three sequences showed extensive homology at the DNA and protein level. Sequence diversity was located in two discrete regions, which are therefore likely to contain epitopes responsible for the antigenic (subtype) heterogeneity of these proteins. Class 1 proteins from all three meningococcal strains were expressed at low levels in E.coli. The apparent molecular weight by SDS-PAGE of the recombinant protein was identical to that of the native protein in the meningococcus, indicating that these proteins are processed and possibly translocated to the E.coli outer membrane.

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Published date: 1989

Identifiers

Local EPrints ID: 458290
URI: http://eprints.soton.ac.uk/id/eprint/458290
PURE UUID: 57fa029d-e7a2-4434-a400-fdf023691afb

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Date deposited: 04 Jul 2022 16:46
Last modified: 23 Jul 2022 00:15

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Contributors

Author: Ann Katherine Barlow

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