Blockade of CD40/CD40L interactions prolongs the survival time of mice with graft-versus-host disease

Abstract

Abstract: AIM: To clone and express human CD40-Ig fusion protein, and further investigate its protective effects on graft-versus-host disease in a murine model. METHODS: Human CD40 gene extracellular region was cloned from Daudi cell line by RT-PCR and inserted into plasmid pIG1, which contains genomic human IgG1 Fc fragment.Then a transient vector containing CD40-Fc fusion gene was constructed and transfected into COS-7 cells.The secreted expression of CD40-Ig fusion protein was detected through indirect sandwich ELISA.In order to obtain plentiful CD40-Ig fusion protein, a constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1, and hence transfecting it into CHO cells.Positive recombinant CHO cells, which secrete CD40-Ig fusion protein, were selected out and cultured on a large scale with serum-free medium, culture supernatant was harvested and purified by protein A affinity chromatography.SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig.Murine acute graft-versus-host disease (GVHD) was induced by intravenous (iv) injection of C57BL/6J (H-2^b) spleen cells into sub-lethally irradiated BALB/c (H-2^d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated. RESULTS: Mammalian expression vectors pIG/40-Ig and p3.1 40-Ig were constructed as described above.Chimeric proteins were expressed in COS-7 cell and CHO cell culture supernatant and confirmed by ELISA and Western blot.SDS-PAGE showed that fusion protein has a disulfide- bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L expressed on Jurkat cell line.In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolongs the mean survival time of mice with GVHD. CONCLUSION: The CD40/CD40L interactions play a pivotal role in the pathogenesis of GVHD, suggesting clinical potential for CD40-Ig in the prevention and treatment of human GVHD.

Key words: biomedical engineering, CD40-Ig, fusion protein, graft-versus-host disease

CLC Number:

R318

LIU He-Zhong, MAO Ning, HOU Chun-Mei, LI Xiu-Sen, TANG Pei-Xian, SHEN Bei-Fen. Blockade of CD40/CD40L interactions prolongs the survival time of mice with graft-versus-host disease[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2003, 8(1): 6-10.

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