Expression of a male accessory gland peptide of Leptinotarsa decemlineata in insect cells infected with a recombinant baculovirus.
The male accessory glands (MAGs) of Leptinotarsa decemlineata produce an 8kDa peptide, designated Led-MAGP, that is recognized by monoclonal antibody MAC-18. The site of synthesis, amino acid sequence and the gene encoding this peptide have been documented ([Smid and Schooneveld, 1992][Smid et al., 1997]). The primary structure is homologous to the N-terminal hexa-repeat section of the chicken prion protein ([Harris et al., 1991]). The biological function of the Led-MAGP has yet to be determined. For further research, large amounts of Led-MAGP is required, both for the production of a more specific antiserum, as well as for application in bio-assays. This paper describes the expression of Led-MAGP in insect cells infected with recombinant baculovirus, and the production of a polyclonal antibody against this recombinant peptide. The peptide was expressed under the control of the polyhedrin promotor. The resulting product was HPLC-purified, and analysis on Western blots immuno-labelled with MAC-18 confirmed that the correct peptide was produced. Purified recombinant peptide was also analyzed by Edman degradation and mass spectrometry; this indicated that it was N-terminally blocked and that the methionine residue at position 7 was oxidized. Large scale production resulted in the formation of aggregations of Led-MAGP, nevertheless a substantial proportion remained in a soluble state and could be harvested. A polyclonal antiserum encoded #87 was produced against recombinant Led-MAGP and its specificity was tested on Western blots of authentic peptide and on LM and EM sections of MAGs. All labelling results were equal to those obtained after MAC-18 labelling. However, antiserum #87 proved to be superior compared to MAC-18, since it recognizes the MAG peptide in normally fed, sexually active males, whereas MAC-18 labelling can only be accomplished after 7 days of starvation of the males. Therefore, the new antiserum #87 enables us to study the transfer dynamics of the Led-MAGP on histological sections.
Vlakb
J M.
JM
Departments of Entomologya and Virologyb, Wageningen Agricultural University, Binnenhaven 7, NL-6709 PD, Wageningen, The Netherlands
Putb
B
B
Desernob
M L.L.G.
ML
Schoonevelda
H
H
Smida
H M.
HM