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Use of genetic immunization to generate a high-level antibody against rat dicarboxylate transporter

  • Nephrology - Original Paper
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Abstract

Background

Rat dicarboxylate transporter (SDCT1), expressed in renal tubular epithelial cells, plays a key role in regulating blood and urinary citrate level by reabsorbing citrate from the lumen. Antibodies against this transporter are very important for investigating its expression and function. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages compared with current methods. This study aimed, by genetic immunization, to produce a high-specificity antibody against SDCT1.

Methods

We fused a high-antigenicity fragment of SDCT1 to the plasmid pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin. Mice were immunized by gene-gun immunization with recombinant plasmid and two other adjuvant plasmids that express granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, respectively. The titer of the antibody was detected by enzyme-linked immunosorbent assay (ELISA). Specificity of the antibody was identified with SDCT1 native protein in rat kidney by Western blot analysis and immunohistochemistry, and with SDCT1 protein expressed on Xenopus oocytes plasma membranes by immunofluorescence.

Results

ELISA measurements showed that the antibody titer was 1:32,000. The native protein of SDCT1 in rat kidney can be recognized by this antibody with Western blot analysis and immunohistochemistry. Immunofluorescence showed that this antibody also recognized SDCT1 protein targeted to Xenopus oocytes plasma membranes into which SDCT1 full-length cRNA was injected.

Conclusion

Generation of a high-specificity immunoglobulin G antibody against SDCT1 by genetic immunization has provided an important tool for the study of citrate transport.

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References

  1. Pajor AM (2006) Molecular properties of the SLC13 family of dicarboxylate and sulfate transporters. Pflugers Arch 451:597–605

    Article  PubMed  CAS  Google Scholar 

  2. Chen XZ, Shayakul C, Berger UV et al (1998) Characterization of a rat Na+-dicarboxylate cotransporter. J Biol Chem 273:20972–20981

    Article  PubMed  CAS  Google Scholar 

  3. Chen X, Tsukaguchi H, Chen XZ et al (1999) Molecular and functional analysis of SDCT2, a novel rat sodium-dependent dicarboxylate transporter. J Clin Invest 103:1159–1168

    Article  PubMed  CAS  Google Scholar 

  4. Pak CY (1994) Citrate and renal calculi: an update. Miner Electrolyte Metab 20:371–377

    PubMed  CAS  Google Scholar 

  5. Jenkins AD, Dousa TP, Smith LH (1985) Transport of citrate across renal brush border membrane: effects of dietary acid and alkali loading. Am J Physiol 249:F590–F595

    PubMed  CAS  Google Scholar 

  6. Wu D, Chen XM, Ye YZ et al (2000) Expression of citrate transporter mRNA in the kidneys of rats with metabolic acidosis (Chinese). Sheng Li Xue Bao 52:55–58

    PubMed  CAS  Google Scholar 

  7. Elbashir MI, Nilson BH, Akesson P et al (1990) Antibody response in immunized rabbits measured with bacterial immunoglobulin-binding proteins. J Immunol Methods 135:171–179

    Article  PubMed  CAS  Google Scholar 

  8. Pizza M, Scarlato V, Masignani V et al (2000) Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287:1816–1820

    Article  PubMed  CAS  Google Scholar 

  9. Wang W, Guo G, Tang J et al (2000) Stimulatory activity of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptors. Chin Med J 113:867–871

    PubMed  CAS  Google Scholar 

  10. Field CM, Oegema K, Zheng Y et al (1998) Purification of cytoskeletal proteins using peptide antibodies. Methods Enzymol 298:525–541

    Article  PubMed  CAS  Google Scholar 

  11. Babiuk LA, van Drunen Littel-van den Hurk S, Babiuk SL (1999) Immunization of animals: from DNA to the dinner plate. Vet Immunol Immunopathol 72:189–202

    Article  PubMed  CAS  Google Scholar 

  12. Tang DC, DeVit M, Johnston SA (1992) Genetic immunization is a simple method for eliciting an immune response. Nature 356:152–154

    Article  PubMed  CAS  Google Scholar 

  13. Ghochikyan A, Vasilevko V, Petrushina I et al (2003) Generation and characterization of the humoral immune response to DNA immunization with a chimeric beta-amyloid-interleukin-4 minigene. Eur J Immunol 33:3232–3241

    Article  PubMed  CAS  Google Scholar 

  14. Trinchieri G (1998) Immunobiology of interleukin-12. Immunol Res 17:269–278

    Article  PubMed  CAS  Google Scholar 

  15. Barouch DH, Letvin NL, Seder RA (2004) The role of cytokine DNAs as vaccine adjuvants for optimizing cellular immune responses. Immunol Rev 202:266–274

    Article  PubMed  CAS  Google Scholar 

  16. Baca-Estrada ME, Ewen C, Mahony D et al (2002) The haemopoietic growth factor, Flt3L, alters the immune response induced by transcutaneous immunization. Immunology 107:69–76

    Article  PubMed  CAS  Google Scholar 

  17. Chambers RS, Johnston SA (2003) High-level generation of polyclonal antibodies by genetic immunization. Nat Biotechnol 21:1088–1092

    Article  PubMed  CAS  Google Scholar 

  18. Zheng X, Xie L, Qin J et al (2008) Effects of wortmannin on phosphorylation of PDK1, GSK3-beta, PTEN and expression of Skp2 mRNA after ischemia/reperfusion injury in the mouse kidney. Int Urol Nephrol 40:185–192

    Article  PubMed  CAS  Google Scholar 

  19. Sun W, Hu Y, Gong J et al (2005) Identification of beta-lactamase inhibitory peptide using yeast two-hybrid system. Biochemistry (Mosc) 70:753–760

    Article  CAS  Google Scholar 

  20. Eslamifar A, Hamkar R, Ramezani A et al (2007) Hepatitis G virus exposure in dialysis patients. Int Urol Nephrol 39:1257–1263

    Article  PubMed  Google Scholar 

  21. Hosoyamada M, Ichida K, Enomoto A et al (2004) Function and localization of urate transporter 1 in mouse kidney. J Am Soc Nephrol 15:261–268

    Article  PubMed  CAS  Google Scholar 

  22. Zhao J, Tramontano A, Makker SP (2007) Albumin-stimulated TGFbeta-1 in renal tubular cells is associated with activation of MAP kinase. Int Urol Nephrol 39:1265–1271

    Article  PubMed  CAS  Google Scholar 

  23. Kaygusuz G, Tulunay O, Baltaci S et al (2007) Microvessel density and regulators of angiogenesis in malignant and nonmalignant prostate tissue. Int Urol Nephrol 39:841–850

    Article  PubMed  CAS  Google Scholar 

  24. Enomoto A, Kimura H, Chairoungdua A et al (2002) Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature 417:447–452

    PubMed  CAS  Google Scholar 

  25. Wang Z, Yuan Z, Matsumoto M et al (2005) Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice. Biochem Biophys Res Commun 327:130–135

    Article  PubMed  CAS  Google Scholar 

  26. Zheng LY, Mou L, Lin S et al (2005) Enhancing DNA vaccine potency against hantavirus by co-administration of interleukin-12 expression vector as a genetic adjuvant. Chin Med J 118:313–319

    PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Dr. Ross Chambers for providing pBQAP-TT, pCMVi-GMCSF, and pCMVi-FIT3L plasmids, Dr. Xing-Zhen Chen for providing pBluescript vector containing SDCT1 full-length cDNA, and Dr. Xuesong Liu for providing anti-mouse Ig-subclass-specific HRP-conjugated secondary antibodies. This work was partly supported by a grant (XJGX0736L04) from Xijing Hospital, Xi’an, China.

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Authors and Affiliations

  1. Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, P.R. China

    Guoshuang Xu & Xiaowei Liu

  2. Emergency Department, Xi’an Children’s Hospital, Xi’an, 710003, P.R. China

    An Liu

Authors
  1. Guoshuang Xu
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  2. An Liu
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  3. Xiaowei Liu
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Corresponding author

Correspondence to Guoshuang Xu.

Additional information

G. Xu and A. Liu contributed equally to this work.

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Xu, G., Liu, A. & Liu, X. Use of genetic immunization to generate a high-level antibody against rat dicarboxylate transporter. Int Urol Nephrol 41, 171–178 (2009). https://doi.org/10.1007/s11255-008-9432-x

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  • DOI: https://doi.org/10.1007/s11255-008-9432-x

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