A common variant associated with asthma, interleukin 13 R130Q, promotes the production of IgE.
Interleukin (IL)-13 plays an important role in the pathogenesis of asthma. A polymorphic variant of human IL-13 R130Q, results in substitution of an arginine with a glutamine was shown to be associated with asthma in Chinese Han nationality. We examined the functional consequences of this variant in vitro to investigate whether this variant enhanced functional activity compared with wild type IL-13. The wild-type and mutant IL-13 genes were amplified from the plasmid of pET22b-hIL-13 by PCR and site-directed mutagenesis PCR. Both the PCR product and the vector pET28a(+) were digested by the NdeI and BamHI. Then the PCR product was cloned in the prokaryotic expression vector of pET28a(+). The plasmids were constructed and transformed into E. coli BL21(DE3). The positive clones were selected, and tested by sequencing. Peripheral blood mononuclear cells (PBMCs) from healthy participants were isolated and cultured with increasing concentrations of recombinant WT IL-13 and IL-13 R130Q. IgE was detected with ELISA kit in the supernatants. Recombinant WT IL-13 and IL-13 R130Q were successfully expressed into the prokaryotic expression system and their biological activity was consistent with standard protein. Our results show that IL-13 R130Q is more active than WT IL-13 in inducing hydrocortisone-dependent IgE synthesis. There were statistical significances between them. IgE induction by physiologic concentrations was obviously increased. IL-13 R130Q has increased activity compared with wild type IL-13 in vitro. And IL-13 R130Q may be used for new target of asthma for diagnosis and therapy in the future.
Department of Pediatrics, Xin Hua Hospital affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China.
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Chu
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Hua
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Liu
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Bao
D000328:Adult; D001249:Asthma / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D056726:Genetic Association Studies; D005822:Genetic Vectors; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology*; D018793:Interleukin-13 / Q000235:genetics* / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008297:Male; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055815:Young Adult
D018793:Interleukin-13; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D006854:Hydrocortisone