[Cloning and expression of human interleukin-21 cDNA in E.coli].
OBJECTIVE
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.


CONCLUSIONS
The rhIL-21 with bioactivity was obtained, which lays the foundation for study of its function.
Zhang
Sheng-quan
SQ
Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei 230032, China.
Chen
Bing
B
Luo
Xin
X
Xu
Cong-zhen
CZ
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D049109:Cell Proliferation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D007378:Interleukins / Q000096:biosynthesis / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology; D000098546:Interleukin-21
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D018076:DNA, Complementary; D007378:Interleukins; D011994:Recombinant Proteins; D000098546:Interleukin-21