Related Experiment Video
Updated: Aug 23, 2026
Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Published on: January 22, 2014
[Cloning and expression of human interleukin-21 cDNA in E.coli]
Sheng-quan Zhang1, Bing Chen, Xin Luo
1Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei 230032, China.
Aim:
To clone full length cDNA of human interleukin-21 (IL-21) and express it in E.coli.
Methods:
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.
Results:
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.
Conclusion:
The rhIL-21 with bioactivity was obtained, which lays the foundation for study of its function.
