新型人白细胞介素-13在大肠杆菌中的表达及生物活性分析

目的 :在大肠杆菌中表达人白细胞介素 13的新型拼接体蛋白。方法 :用植物凝集素 (PHA)和刀豆凝集素 (ConA)共刺激人Jurkat细胞 ,通过半巢式RT PCR扩增在 98位缺失Gln、不含信号肽的人IL 13新拼接体基因 ,并克隆入 pBV2 2 0 ,构建 pBVIL 13表达载体 ,经温度诱导后在大肠杆菌DH5α中表达新型人IL 13蛋白。表达产物经S 2 0 0纯化、复性后 ,用TF 1细胞进行MTT比色测定其活性。结果 :成功地分离了人IL 13新拼接体基因 ,并在大肠杆菌中表达。分离纯化的新型IL 13蛋白 ,其比活性为 1.6× 10 6U/mg。结论 :获得具有生物学活性的新型人IL 13细胞因子 ,为用其治疗肿瘤和脓毒症提供了新的手段

Expression of a novel human interleukin-13 in E.coli and its biological activity assay

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.