Journal of Clinical Immunology

, Volume 25, Issue 5, pp 445–451 | Cite as

Primary Immune Effects of Eukaryotic Expression Plasmids Encoding Two Hyperactive Mutants of Human Soluble B Lymphocyte Stimulator

  • Guangyu Chen
  • Hongwu Du
  • Zhengjian Zhang
  • Shanyun Peng
  • Donggang Xu
  • Jiaxi Wang


B lymphocyte stimulator (BLyS), a ligand belonging to the tumor necrosis factor (TNF) family, plays a critical role in regulating survival and activation of peripheral B cell populations during humeral immune responses. Among the TNF family members, BLyS is unique in that it contains an unpaired Cys residue (Cys146) at the corresponding position where some other members have about 37.5% (6/16) Ala or 37.5% (6/16) Val. Here, with eukaryotic expression vector pcDNA3.1(−), we mutated Cys146 to Ala or Val and constructed two mutant eukaryotic expression plasmids of the human soluble BLyS, pcDNA3.1BY-A and pcDNA3.1BY-V. Following repetitive subcutaneous injection of these expression plasmids in BALb/C mice, the wild-type and mutant BLyS proteins were detectable in the blood of treated animals over several weeks. In addition, the expression of these proteins induced specific IgG but not IgM responses. The implications of the results are discussed.


B cell proliferation BLyS cytokine immunoglobulin secretion mutation 


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  1. 1.
    Lui VW, He Y, Falo L, Huang L: Systemic administration of naked DNA encoding interleukin 12 for the treatment of human papillomavirus DNA-positive tumor. Hum Gene Ther 13:177, 2002CrossRefPubMedGoogle Scholar
  2. 2.
    Ficara F, Superchi DB, Hernandez RJ, Mocchetti C, Carballido-Perrig N, Andolfi G, Deola S, Colombo A, Bordignon C, Carballido JM, Roncarolo MG, Aiuti A: IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential. Mol Ther 10:1096, 2004CrossRefPubMedGoogle Scholar
  3. 3.
    Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, Soppet D, Charters M, Gentz R, Parmelee D, Li YL, Galperina O, Giri J, Roschke V, Nardelli B, Carrell J, Sosnovtseva S, Ruben SM, Olsen HS, Fikes J, Hilbert DM: BLyS: A member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260, 1999CrossRefPubMedGoogle Scholar
  4. 4.
    Mukhopadhyay A, Ni J, Zhai YF, Yu GL, Aggarwal BB: Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J Biol Chem 274:15978, 1999CrossRefPubMedGoogle Scholar
  5. 5.
    Schneider P, MacKay F, Steiner V, Hofmann K, Bodmer JL, Holler N, Ambrose C, Lawton P, Bixler S, Acha-Orbea H, Valmori D, Romero P, Werner-Favre C, Zubler RH, Browning JL, Tschopp J: BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med 189:1747, 1999CrossRefPubMedGoogle Scholar
  6. 6.
    Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, Tschoop J, Browning JL: Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med 190:1697, 1999CrossRefPubMedGoogle Scholar
  7. 7.
    Khare SD, Sarosi I, Xia XZ, McCabe S, Miner K, Solovyev I, Hawkins N, Kelley M, Chang D, Van G, Ross L, Delaney J, Wang L, Lacey D, Boyle WJ, Hsu H: Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci USA 97:3370, 2000CrossRefPubMedGoogle Scholar
  8. 8.
    Melchers F: Actions of BAFF in B cell maturation and its effects on the development of autoimmune disease. Ann Rheum Dis 62(Suppl 2):II25, 2003CrossRefPubMedGoogle Scholar
  9. 9.
    Schneider P, Tschopp J: BAFF and the regulation of B cell survival. Immunol Lett 88:57, 2003CrossRefPubMedGoogle Scholar
  10. 10.
    Mackay F, Browning JL: BAFF: A fundamental survival factor for B cells. Nat Rev Immunol 2:465, 2002CrossRefPubMedGoogle Scholar
  11. 11.
    Schiemann B, Gommerman JL, Vora K, Cachero TG, Shulga-Morskaya S, Dobles M, Frew E, Scott ML: An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293:2111, 2001Google Scholar
  12. 12.
    Rahman ZS, Rao SP, Kalled SL, Manser T: Normal induction but attenuated progression of germinal center responses in BAFF and BAFF-R signaling-deficient mice. J Exp Med 198:1157, 2003CrossRefPubMedGoogle Scholar
  13. 13.
    Parry TJ, Riccobene TA, Strawn SJ, Williams R, Daoud R, Carrell J, Sosnovtseva S, Miceli RC, Poortman CM, Sekut L, Li Y, Fikes J, Sung C: Pharmacokinetics and immunological effects of exogenously administered recombinant human B lymphocyte stimulator (blys) in mice. J Pharmacol Exp Ther 296:396, 2001PubMedGoogle Scholar
  14. 14.
    Stewart DM, McAvoy MJ, Hilbert DM, Nelson DL: B lymphocytes from individuals with common variable immunodeficiency respond to B lymphocyte stimulator (BLyS protein) in vitro. Clin Immunol 109:137, 2003CrossRefPubMedGoogle Scholar
  15. 15.
    Bodmer JL, Meier P, Tschopp J, Schneider P: Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 275:20632, 2000CrossRefPubMedGoogle Scholar
  16. 16.
    Do RKG, Hatada E, Lee H, Tourigny MR, Hilbert D, Chen-Kiang S: Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response. J Exp Med 192:953, 2000CrossRefPubMedGoogle Scholar
  17. 17.
    Chen G, Zou M, Peng S, Wang J: Cloning, expression, and activity determination of the recombinant human soluble B lymphocyte stimulator and its two mutants. Acta Biochim Biophys Sin 34:731, 2002PubMedGoogle Scholar
  18. 18.
    Chen G, Peng S, Zou M, Wang J: Construction and function of two cysteine146-mutants with high activity, derived from recombinant human soluble B lymphocyte stimulator. J Biochem 136:74, 2004Google Scholar
  19. 19.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989Google Scholar
  20. 20.
    Vieillard V, Cremer I, Lauret E, Rozenbaum W, Debre P, Autran B, De Maeyer E: Interferon beta transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens. Proc Natl Acad Sci USA 94:11595, 1997CrossRefPubMedGoogle Scholar
  21. 21.
    Martinenghi, S, Cusella De Angelis, G, Biressi, S, Amadio, S, Bifari, F, Roncarolo, MG, Bordignon, C, Falqui, L: Human insulin production and amelioration of diabetes in mice by electrotransfer-enhanced plasmid DNA gene transfer to the skeletal muscle. Gene Ther 9:1429, 2002CrossRefPubMedGoogle Scholar
  22. 22.
    Demers GW, Sugarman BJ, Beltran JC, Westreich LN, Ahmed CM, Lau JY, Hong Z, Lanford RE, Maneval DC: Interferon-alpha2b secretion by adenovirus-mediated gene delivery in rat, rabbit, and chimpanzee results in similar pharmacokinetic profiles. Toxicol Appl Pharmacol 180:36, 2002CrossRefPubMedGoogle Scholar
  23. 23.
    Ju W, Liu J, Xiao W, Liu M, Qu X: Construction of a eukaryotic expression system of HSP65 gene from Mycobacterium tuberculosis, and anti-HSP65 IgG produced in mice. J Med Microbiol 54:3, 2005CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Guangyu Chen
    • 1
  • Hongwu Du
    • 2
  • Zhengjian Zhang
    • 3
  • Shanyun Peng
    • 1
  • Donggang Xu
    • 1
  • Jiaxi Wang
    • 1
    • 4
  1. 1.Laboratory of Molecular GeneticsBeijing Institute of Basic Medical SciencesBeijingP. R. China
  2. 2.Department of Biological Sciences and BiotechnologyTsinghua UniversityBeijingP. R. China
  3. 3.The Pennsylvania State UniversityPhiladelphia
  4. 4.Laboratory of Molecular GeneticsBeijing Institute of Basic Medical SciencesBeijingP. R. China

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