重组人IL-29的原核表达、纯化及活性分析

Prokaryotic expression, purification and activity analysis of recombinant human interleukin-29

目的:在大肠杆菌中表达具有活性的重组人白细胞介素-29(IL-29).方法:用PCR技术扩增人IL-29成熟蛋白的编码序列,克隆入原核表达载体pET-44Ek/LIC中,构建融合表达载体pET-44Ek/LIC-IL-29,转化大肠杆菌BL21(DE3),以IPTG诱导表达IL-29融合蛋白,经镍柱亲和层析纯化,然后用肠激酶消化除去N端融合标签得到完全天然状态的重组人IL-29,经N-端氨基酸测序鉴定纯化的IL-29,用细胞病变抑制法测定其活性.结果:成功构建了表达载体pET-44Ek/LIC-IL-29,DNA序列测定结果与预期结果一致.在30℃培养条件下,IPTG诱导表达的IL-29融合蛋白占菌体蛋白总量43%左右,并且大部分以可溶形式存在.纯化后的融合蛋白经肠激酶切割和回收后,所得目的蛋白(IL-29)纯度大于96%,该蛋白N-端序列与理论值一致,其抗病毒活性与IFN-α2b相当.结论:获得了具有生物学活性的重组人IL-29.

AIM ： To express recombinant human interleukin-29 （IL-29） with biological activity in E. coll. METHODS： The cDNA fragment coding for mature IL-29 protein was amplified by PCR and cloned into vector pET-44 Ek/LIC to construct fusion expression vector pET-44 Ek/LIC-IL-29. After pET-44 Ek/LIC- IL-29 was transformed into E. coli BI221 （ DE3 ）, the bacteria were induced by IPTG. The expressed IL-29 fusion protein was purified by Ni-NTA affinity chromatography and the fusion tag was removed from IL-29 fusion protein by cleavage with enterokinase. The purified IL-29 was subjected to N-terminal sequencing. The antiviral activity of IL-29 was detected by cytopathic effect reduction assay. RESULTS： The DNA sequencing showed that the expression vector pET-44 Ek/LIC-IL-29 was constructed successfully. After induced by IPTG, the target protein accounted for 43% of the total bacterial protein and most was expressed in a soluble form in E. coli cultured at 30℃. The purified IL-29 appeared a single band on SDS-PAGE and its purity was more than 96%. The first 10 amino acid sequence of the N-terminus was consistent with the theoretical sequence. The recombinant IL- 29 showed specific antiviral activity that was comparable to the commercially available IFN-α2b preparation. CONCLUSION ： The recombinant human IL-29 with biological activity has been obtained.