Production of Monoclonal Antibodies of IgM Type in DG44 Cells

Автор: M.A. Ershler, and N.I. Olovnikova

Страница: 24-34

Production of Monoclonal Antibodies of IgM Type in DG44 Cells

Biotekhnologiya, 2014, N 2, P. 24-34

UDC 577.21

Section:  “Producers, Biology, Selection, and Gene Engineering”

M.A. Ershler ^*,  and  N.I. Olovnikova

The Hematological Research Center, Ministry of Healthcare of RF,  125167, Moscow Russia

e-mail:  ershler@yandex.ru

Two-bicistronic vectors for the production of recombinant IgM monoclonal antibodies in the DG44 DHFR-negative cell line have been designed. We used tandem vectors in which one bicistronic unit encoded the immunoglobulin light chain and DHFR, whereas the other encoded the heavy chain and EGFP. The constructs structure presumes that green colored cells that survived selection would be capable of producing both immunoglobulin chains. We found out that the agglutinating IgM antibodies could be secreted in the absence of J peptide. It was shown that germinal leader peptide plays a key role in the expression of the genes for the light and heavy chains. A comparison of the chromatin regulatory elements demonstrated that construct-flanking 2xHS4 insulators stabilized the biosynthesis of the recombinant antibodies, whereas the 5'-MARLyz matrix attachment region proved to be less efficient. The strategy of obtaining of a DG44-based producer cell line should include the following consecutive steps: selection on the medium without nucleosides → amplification of the inserted gene → cloning of transfectants → selection of high-productive clones. An attempt to clone before amplification and amplify individual clones failed to result in effective producers. Cloning on a medium without selection pressure allows more adequate assessing of the stability of the antibody production.

Key words:  amplification,  DG44,  IgM,  monoclonal antibodies,  vector.

17.03.2015, 1674 просмотра.