人白细胞介素-6重组蛋白的制备

Preparation of human interleukin-6 recombinant protein

目的利用大肠杆菌原核表达系统制备人白细胞介素-6(IL-6)重组蛋白,并对蛋白进行纯化,为后期制备IL-6标准物质和质控品提供候选物质基础。方法以现有IL-6原核表达质粒6His-IL-6-pET-28a(+)为模板,通过PCR扩增目的片段,并在其N端添加His标签。通过酶切连接的方式,将该段基因克隆至pRSFDuet载体,随后转化BL21感受肽获得表达菌株。利用异丙基硫代半乳糖苷(IPTG)进行诱导,得到IL-6重组蛋白,利用镍柱亲和层析技术对蛋白进行纯化,并比对两个表达菌株的蛋白表达量,选择高表达菌株进行后续实验。结果成功构建6His-IL-6-pRSFDuet原核表达质粒,根据蛋白表达比对情况选择6His-IL-6-pET-28a(+)质粒进行后续实验,确定最适IPTG诱导浓度(0.2 mmol/L)、诱导温度(20℃)、诱导时间(20 h)等条件,在BL21中高效表达重组IL-6蛋白,纯化后的蛋白不仅可通过临床检测,且稀释倍数与检测结果之间呈线性关系。结论成功获得低成本、高浓度、高纯度的IL-6重组蛋白,为后续IL-6标准物质和质控品的制备奠定了基础。

Objective To express and purify human interleukin-6(IL-6)recombinant protein via prokaryotic expression system,which provides a candidate material for the preparation of IL-6 reference and quality control materials at a later stage.Methods The target gene segment was amplified by PCR using the existing IL-6 prokaryotic expression plasmid 6His-IL-6-pET-28a(+)as a template and a His-tag was added to its N-terminus.The synthetic segment was cloned into the pRSFDuet prokaryotic expression vector by enzyme digestion and ligation to construct a 6His-IL-6-pRSFDuet recombinant plasmid,and then the plasmid was transformed into E.coli BL21 expressing strain.The expression of recombinant IL-6 protein was induced by isopropyl-β-d-thiogalactoside(IPTG),and the protein was purified by nickel affinity chromatography.The protein expression levels of the two strains were compared,and the one with the higher expression was selected for the subsequent experiments.Results The 6His-IL-6-pRSFDuet prokaryotic expression plasmid was successfully constructed,and the 6His-IL-6-pET-28a(+)plasmid was selected for the subsequent experiments according to the protein expression comparison.After optimizing the expression conditions,it was found that the highest expression of IL-6 recombinant protein could be obtained after 20 h induction with IPTG at a final concentration of 0.2 mmol/L at 20℃.The purified IL-6 protein was detectable in the clinical laboratory,and there was a linear relationship between the dilution ratio and the detection results.Conclusion The low cost,high concentration and high purity of IL-6 recombinant proteins are successfully obtained,which lays the foundation for the subsequent preparation of reference and quality control materials.