Interleukin-21
positive regulation of T cell proliferation
[Cloning and expression of human interleukin-21 cDNA in E.coli].
<h4>Aim</h4>To clone full length cDNA of human interleukin-21 (IL-21) and express it in E.coli.<h4>Methods</h4>Total RNA was isolated from peripheral lymphocyte stimulated with anti-CD3 antibody. 5' and 3' terminal fragments of IL-21 gene (242 bp and 425 bp fragments respectively) were amplified using RT-PCR. The full length IL-21 cDNA was amplified by recombination PCR from the products of RT-PCR. The expression plasmid pET28a(+)-IL21 was constructed by inserting IL-21 cDNA into pET28a(+)and then was transformed into BL21(DE3). Expression of hIL-21 was induced by IPTG at 37 degrees Celsius for 5 h. The target protein was purified through Ni(2+)-chelating Sepharose Fast Flow. Purified rhIL-21 was refolded by using dialysis method. And the bioactivity was detected by MTT on costimulating the T cell proliferation with anti-CD3.<h4>Results</h4>IL-21 was cloned and expressed in E.coli successfully. SDS-PAGE analysis showed the IL-21 was expressed in the form of insoluble inclusion body. The refolded rhIL-21 could stimulate the proliferation of mature human T-cells in the presence of anti-CD3.<h4>Conclusion</h4>The rhIL-21 with bioactivity was obtained, which lays the foundation for study of its function.