Jiang Xiao-feng, Guo Xiao-ying, Wan Li-ping
The Center of Experiment Diagnosis, Second Hospital Affiliated to Harbin Medical University, Harbin 150086, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Sep;22(5):564-7.
To express recombinant wild-type human interleukin-13 (rhIL-13) and mutant interleukin-13 (rhIL-13') in E.coli BL21 (DE3) and get active proteins through purification and renaturation.
IL-13 and IL-13' gene fragments were amplified by PCR and site-directed mutagenesis PCR, respectively and then were inserted into expression vector pET30a(+). Recombinant plasmids were transformed to E.coli BL21 (DE3) and were expressed under IPTG induction. Expressed products were purified through Ni-NTA chromatographic column. The purified proteins were renatured by GSH-GSSG (reduced glutathione, oxidized glutathione) system and dialysis and their bioactivity was detected by MTT colorimetry.
The expressed recombinant proteins existed in the form of inclusion body with relative molecular mass about 17 000. The recombinant proteins with higher purity were obtained after purification. The renatured inclusion bodies were biologically active.
rhIL-13/rhIL-13' with biological activity have been obtained successfully, which lays the foundation for further study on their function.
在大肠杆菌BL21（DE3）中表达重组野生型人白细胞介素-13（rhIL-13）和突变型白细胞介素-13（rhIL-13'），并通过纯化和复性获得活性蛋白。
分别通过PCR和定点突变PCR扩增IL-13和IL-13'基因片段，然后将其插入表达载体pET30a(+)。将重组质粒转化至大肠杆菌BL21（DE3），并在IPTG诱导下表达。表达产物通过Ni-NTA色谱柱纯化。纯化后的蛋白通过谷胱甘肽-氧化型谷胱甘肽（还原型谷胱甘肽，氧化型谷胱甘肽）系统和透析进行复性，其生物活性通过MTT比色法检测。
表达的重组蛋白以包涵体形式存在，相对分子质量约为17 000。纯化后获得了纯度较高的重组蛋白。复性后的包涵体具有生物活性。
已成功获得具有生物活性的rhIL-13/rhIL-13'，为进一步研究其功能奠定了基础。
