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Published on: March 1, 2011
[Expression of a novel human interleukin-13 in E.coli and its biological activity assay]
Da-Dong Wang1, Ji-Ye Li, Jin-Hui Wang
1Department of General Surgery, No.304 Hospital of PLA, Beijing 100037,China.
Aim:
To express a novel Gln98-deleted human interleukin-13 in E.coli.
Methods:
Total RNA was isolated from Jurkat cells costimulated with PHA and ConA. A 358 bp-specific DNA fragment encoding hIL-13 was amplified by semi-nested RT-PCR. DNA sequencing showed that the target DNA was a Gln98-deleted novel splicing of hIL-13. This hIL-13 cDNA and plasmid pBV220 were ligated at BamH I and EcoR I sites to construct the expression vector. After transforming E.coli strain DH5alpha, the expression of the novel splicing hIL-13 gene was induced by shifting culture temperature from 30 degrees Celsius to 42 degrees Celsius. The expression product was then purified by chromatography on Sepharcryl-200 gel column, and the bioactivity was detected by MTT colorimetry on the growth of TF-1 cell line.
Results:
The novel rhIL-13 was expressed in the form of inclusion bodies. After purification and renaturation, the specific activity of the novel rhIL-13 was 1.6x10(6) IU/mg.
Conclusion:
The novel rhIL-13 with biological activity has been obtained, which lays the foundation for treating cancer and septicemia by the cytokine in future.
