Advertisement

Biochemistry (Moscow)

, Volume 73, Issue 9, pp 1025–1030 | Cite as

Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

  • Li Xu
  • Zhouzhou Zhao
  • Jiqun Sheng
  • Chengang Zhu
  • Hui Liu
  • Dahe Jiang
  • Xin Mao
  • Mingxiong Guo
  • Wenxin Li
Article

Abstract

C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.

Key words

complement regulatory proteins internal ribosome entry site (IRES) dicistronic expression vector co-expression hyperacute rejection 

Abbreviations

CMV

cytomegalovirus

CRP

complement regulatory protein

DAF

decay accelerating factor

DMEM

Dulbecco’s modified Eagle’s medium

EMCV

encephalomyocarditis virus

IRES

internal ribosome entry site

MCP

membrane cofactor protein

MTT

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

NHS

normal human serum

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Makrides, S. C. (1998) Pharmacol. Rev., 50, 59–87.PubMedGoogle Scholar
  2. 2.
    Mollnes, T. E., and Fiane, A. E. (2002) Allergy, 72, 75–78.CrossRefGoogle Scholar
  3. 3.
    Brodbeck, W. G., Kuttner-Kondo, L., Mold, C., and Medof, M. E. (2000) Immunology, 101, 104–111.PubMedCrossRefGoogle Scholar
  4. 4.
    Nicholson-Weller, A., Burge, J., Fearon, D. T., Weller, P. F., and Austen, K. F. (1982) J. Immunol., 129, 184–189.PubMedGoogle Scholar
  5. 5.
    Seya, T., Turner, J. R., and Atkinson, J. P. (1986) J. Exp. Med., 163, 837–855.PubMedCrossRefGoogle Scholar
  6. 6.
    Pizzato, M., Franchin, E., Calvi, P., Boschetto, R., Colombo, M., Ferrini, S., and Palu, G. (1998) Gene Ther., 5, 1003–1007.PubMedCrossRefGoogle Scholar
  7. 7.
    Candotti, F., Agbaria, R., Mullen, C. A., Touraine, R., Balzarini, J., Johns, D. G., and Blaese, R. M. (2000) Cancer Gene Ther., 7, 574–580.PubMedCrossRefGoogle Scholar
  8. 8.
    Gaken, J., Jiang, J., Daniel, K., van Berkel, E., Hughes, C., Kuiper, M., Darling, D., Tavassoli, M., Galea-Lauri, J., Ford, K., Kemeny, M., Russell, S., and Farzaneh, F. (2000) Gene Ther., 7, 1979–1985.PubMedCrossRefGoogle Scholar
  9. 9.
    Singh, G., Parker, S., and Hobart, P. (2002) Vaccine, 20, 1400–1411.PubMedCrossRefGoogle Scholar
  10. 10.
    Ghattas, I. R., Sanes, J. R., and Majors, J. E. (1991) Mol. Cell. Biol., 11, 5848–5859.PubMedGoogle Scholar
  11. 11.
    Fan, L., Drew, J., Dunckley, M. G., Owen, J. S., and Dickson, G. (1998) Gene Ther., 5, 1434–1440.PubMedCrossRefGoogle Scholar
  12. 12.
    Martinez-Salas, E. (1999) Curr. Opin. Biotechnol., 10, 458–464.PubMedCrossRefGoogle Scholar
  13. 13.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, New York.Google Scholar
  14. 14.
    Huang, J., Gou, D. M., Zhen, C. Y., Jiang, D. H., Mao, X., and Li, W. X. (2001) FEMS Immunol. Med. Microbiol., 31, 203–209.PubMedCrossRefGoogle Scholar
  15. 15.
    Laemmli, U. K., and Favre, M. (1970) Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  16. 16.
    Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA, 76, 4350–4354.PubMedCrossRefGoogle Scholar
  17. 17.
    Mosmann, T. (1983) J. Immunol. Meth., 65, 55–63.CrossRefGoogle Scholar
  18. 18.
    Menoret, S., Plat, M., Blancho, G., Martinat-Botte, F., Bernard, P., Karam, G., Tesson, L., Renaudin, K., Guillouet, P., Weill, B., Chereau, C., Houdebine, L. M., Soulillou, J. P., Terqui, M., and Anegon, I. (2004) Transplantation, 77, 1468–1471.PubMedCrossRefGoogle Scholar
  19. 19.
    Nagahama, M., Shiraishi, M., Oshiro, T., Taira, K., Sugawa, H., Nozato, E., Nomura, H., Nagamine, M., and Muto, Y. (2002) Transpl. Int., 15, 205–219.PubMedGoogle Scholar
  20. 20.
    Cascalho, M., and Platt, J. (2001) Immunity, 14, 437–446.PubMedCrossRefGoogle Scholar
  21. 21.
    Iwata, K., Seya, T., Ariga, H., and Nagasawa, S. (1994) J. Immunol., 152, 3436–3444.PubMedGoogle Scholar
  22. 22.
    Brodbeck, W. G., Mold, C., Atkinson, J. P., and Medof, M. E. (2000) J. Immunol., 165, 3999–4006.PubMedGoogle Scholar
  23. 23.
    Fukuta, D., Miyagawa, S., Kubo, T., Matsunami, K., Shirasu, A., Hattori, H., and Shirakura, R. (2003) Biochem. Biophys. Res. Commun., 306, 476–482.PubMedCrossRefGoogle Scholar
  24. 24.
    Higgins, P. J., Ko, J. L., Lobell, R., Sardonini, C., Alessi, M. K., and Yeh, C. G. (1997) J. Immunol., 158, 2872–2881.PubMedGoogle Scholar
  25. 25.
    Byrne, G. W., McCurry, K. R., Martin, M. J., McClellan, S. M., Platt, J. L., and Logan, J. S. (1997) Transplantation, 63, 149–155.PubMedCrossRefGoogle Scholar
  26. 26.
    Chen, R. H., Naficy, S., Logan, J. S., Diamond, L. E., and Adams, D. H. (1999) XeNo, 6, 194–200.CrossRefGoogle Scholar
  27. 27.
    Cowan, P. J., Aminian, A., Barlow, H., Brown, A. A., Chen, C. G., Fisicaro, N., Francis, D. M., Goodman, D. J., Han, W., Kurek, M., Nottle, M. B., Pearse, M. J., Salvaris, E., Shinkel, T. A., Stainsby, G. V., Stewart, A. B., and d’Apice, A. J. (2000) Transplantation, 69, 2504–2515.PubMedCrossRefGoogle Scholar

Copyright information

© MAIK Nauka 2008

Authors and Affiliations

  • Li Xu
    • 1
    • 2
  • Zhouzhou Zhao
    • 1
  • Jiqun Sheng
    • 1
  • Chengang Zhu
    • 1
  • Hui Liu
    • 1
  • Dahe Jiang
    • 1
  • Xin Mao
    • 1
  • Mingxiong Guo
    • 1
  • Wenxin Li
    • 1
  1. 1.State Key Laboratory of Virology, College of Life ScienceWuhan UniversityWuhanP. R. China
  2. 2.College of Life Sciences and TechnologyHuazhong University of Science and TechnologyWuhanP. R. China

Personalised recommendations