摘要
目的原核表达人CC趋化因子配体17(CC chemokine ligand 17,CCL17)重组蛋白,纯化并分析其趋化活性。方法根据GenBank中登录的人CCL17基因序列(NM002987.2)设计特异性引物,扩增人静脉全血CCL17基因,将其PCR产物与pCold-TFM载体连接构建重组表达质粒pCold-TFM-CCL17,转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达;表达产物经镍亲和层析,酶切移除融合蛋白的6×His-TF标签后,进行离子交换亲和层析纯化;凝胶过滤层析分析其聚合特性,分光光度法分析蛋白浓度,应用表达CCL17受体的Hut78细胞进行趋化试验。结果经双酶切及测序鉴定证明,重组表达质粒pCold-TFM-CCL17构建正确;融合蛋白以可溶性形式表达;纯化的人CCL17重组蛋白相对分子质量约为11000,纯度达95%以上,其蛋白峰呈均匀、对称的单个色谱峰,色谱峰面积占总曲线面积95%以上,蛋白表达量≥6 mg/L,其可趋化Hut78细胞发生迁移。结论成功表达了可溶性、高纯度、高产量、具备一定趋化活性的人CCL17重组蛋白,为其工业化生产及科研应用提供了参考。
Objective To express recombinant human CC chemokine ligand 17(CCL17)in prokaryotic cells,purify the expressed product and analyze its chemotactic activity.Methods Specific primers were designed according to the human CCL17 gene sequence(NM002987.2)in GenBank,based on which human intravenous blood CCL17 gene was amplified by PCR and inserted into vector pCold-TFM.The constructed recombinant plasmid pCold-TFM-CCL17 was transformed to competent E.coli BL21(DE3)and induced by IPTG.The expressed product was purified by nickel affinity chromatography,and further purified by ion exchange chromatography after the tag 6×His-TF was removed by digestion with restriction enzyme.The polymerization characteristics was analyzed by gel filtration chromatography,while the protein concentration by spectrophotometry.The chemotaxis test was performed by using Hut78 cells expressing CCL17.Results Restriction analysis and sequencing proved that recombinant plasmid pCold-TFM-CCL17 was constructed correctly.The fusion protein was expressed in a soluble form.The purified human CCL17,with a relative molecular mass of about11000,reached a purity of more than 95%,of which the single protein peak was even and symmetrical.The area under the chromatographic peak accounted for more than 95%of total area under the curve.The expressed protein,at a concentration of not less than 6 mg/L,showed chemotactic activity to the migration of Hut78 cells.Conclusion Soluble recombinant human CCL17 protein at high purity and yield,with a certain chemotactic activity,was successfully expressed,which provided a reference for industrial production and application in research of the protein.
作者
曹拓
魏准
彭湘明
CAO Tuo;WEI Zhun;PENG Xiang-ming(Department of Clinical Laboratory,Guangzhou Red Cross Hospital,Medical College,Jinan University,Guangzhou 510220,Guangdong Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2020年第10期1128-1133,1142,共7页
Chinese Journal of Biologicals
关键词
人CC趋化因子配体17基因
原核细胞
基因表达
纯化
趋化活性
分子伴侣
触发因子
Human CC chemokine ligand 17(CCL17)gene
Prokaryotic cells
Gene expression
Purification
Chemotactic activity
Molecular chaperone
Trigger factor
