摘要
目的:制备链亲合素(SA)连接的MIP-3α融合蛋白MIP-3α-SA,并研究其生物学活性及功能。方法:构建MIP-3α-SA-pET21重组表达载体,在大肠杆菌中诱导表达MIP-3α-SA融合蛋白,经镍金属螯合(Ni-NTA)层析纯化,尿素梯度透析复性,聚丙烯酰胺凝胶电泳法(SDS-PAGE)、Western blot及银氨染色法鉴定融合蛋白;通过体外淋巴细胞趋化实验验证其趋化活性,流式细胞术分析其对已生物素化的小鼠前列腺癌RM-1细胞的表面锚定修饰效率。结果:MIP-3α-SA融合蛋白可在大肠杆菌Rosetta(DE3)中实现高效诱导表达,表达量占细菌总蛋白量的30%。经镍柱亲和层析纯化后该融合蛋白纯度达到95%。经尿素梯度复性后,验证该融合蛋白具有双重活性,即:MIP-3α介导的对人淋巴细胞的趋化作用且呈剂量依赖性,及SA介导的高效结合至表面已生物素化的小鼠前列腺癌RM-1细胞的功能(表面锚定修饰效率大于95%)。结论:MIP-3α-SA双功能融合蛋白具有双重活性,为MIP-3α表面锚定修饰的肿瘤细胞疫苗的研制奠定基础。
Objective: To prepare the streptavidin-tag MIP-3α fusion protein MIP-3α-SA, and to study its biological function and activity. Methods: The recombinant expression vector of MIP-3α-SA-pET21 was constructed and induced to express MIP-3α-SA fusion protein in Escherichia coli. MIP-3α-SA fusion protein was purified by the Ni-NTA affinity chromatography and refolded by gradient dialysis of urea. The fusion protein was identified by amide gel electrophoresis (SDS-PAGE), Western blot and silver ammonia staining. In vitro lymphocyte chemotaxis assays was used to detect the chemotactic activity of the fusion protein, and flow cytometry (FCM) analysis was performed to detect the modification efficiency of the fusion protein on biotin RM-1 cell surface anchoring. Results: The MIP-3α-SA fusion protein expression was induced with high efficiency in E. coli Rosetta (DE3), which accounted for 30% of the total bacterial protein. The purity of the fusion protein was 95% after purification by nickel column affinity chromatography. After renaturation by urea gradient, the fusion protein was verified to have dual activity, namely: MIP-3α-SA stimulated human lymphocyte chemotactic activity has a dose dependent manner and SA-mediated efficient binding to the surface has biotinylated RM-1 cell function (modification of the surface anchoring efficiency of more than 95%). Conclusion: The MIP-3α-SA bifunctional fusion protein has dual activity, which lays the foundation for the research and development of MIP-3α surface-modified tumor cell vaccine.
作者
宋志纯
戴数
王朋
高基民
SONG Zhichun;DAI Shu;WANG Peng;GAO Jimin(Department of Laboratory Medicine, Lishui People’s Hospital, Lishui 323000, China;School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China)
出处
《温州医科大学学报》
CAS
2019年第5期360-366,共7页
Journal of Wenzhou Medical University
基金
丽水市科技计划项目(2014JYZB13)
关键词
巨噬细胞炎性蛋白3α
链亲和素
融合蛋白
锚定修饰
macrophage inflammatory protein-3α
streptavidin fusion protein
anchoring modification
