融合铁蛋白表达、纯化及肿瘤摄取分析

Expression, purification, and tumor uptake of fusion ferritin

摘要:

铁蛋白由于其靶向性、可逆性自组装、高生物相容性及易被修饰等优点，被认为是一种理想的递送系统。本研究旨在表达纯化3种融合铁蛋白，对其进行鉴定，并探究其肿瘤靶向性。合成3种融合铁蛋白基因并克隆至原核表达载体，使用镍柱进行亲和层析纯化重组蛋白，通过非变性聚丙烯酰胺凝胶电泳(native-PAGE)、Western blotting、圆二色谱对融合铁蛋白进行鉴定。使用异硫氰酸荧光素酯(fluorescein 5-isothiocyanate,FITC)与融合铁蛋白进行反应，利用激光共聚焦扫描显微镜进行体外肿瘤细胞靶向。同时将荧光染料胺活性琥珀酰亚胺酯(sulfo-cyanine7,Cy7-SE)与融合铁蛋白进行反应尾静脉注射入黑色素瘤小鼠进行体内肿瘤成像，探究融合铁蛋白的肿瘤靶向性。结果显示，本研究构建的融合铁蛋白使用异丙基-β-d-硫代半乳糖苷(isopropylthio-β-d-galactoside,IPTG)进行诱导表达可获得约21 kDa的可溶性融合铁蛋白，通过镍柱进行亲和层析纯化获得纯度较高的融合铁蛋白。Western blotting检测重组蛋白能被相应抗体识别。Native-PAGE、圆二色谱鉴定目的蛋白为具有α螺旋的多聚体。通过体内外肿瘤摄取实验，证明了融合铁蛋白能够被肿瘤细胞和肿瘤组织摄取。本研究成功表达纯化融合铁蛋白并进行鉴定，验证了其在体内外的肿瘤摄取情况，为铁蛋白在生物医药领域的应用奠定了基础。

Abstract:

Ferritin is considered as an ideal delivery system due to its precise targeting, reversible self-assembly, high biocompatibility, and easy modification. this study aims to express, purify, and identify three fusion ferritin proteins, and explore their tumor targeting. Three fusion ferritin genes were synthesized and cloned into prokaryotic expression vectors, and the recombinant proteins were purified by affinity chromatography with nickel columns. The fusion ferritin proteins were identified by native polyacrylamide gel electrophoresis (native-PAGE), Western blotting, and circular dichroism. Fluorescein 5-isothiocyanate (FITC) was used to react with fusion ferritin, and confocal laser scanning microscopy was employed to evaluate the tumor targeting of fusion ferritin. The reaction system of sulfo-cyanine7 (Cy7-SE) with fusion ferritin was injected into the tail vein of melanoma mice for in vivo tumor imaging to explore the tumor targeting of fusion ferritin. The results showed that soluble fusion ferritin proteins of about 21 kDa were expressed under the induction by isopropylthio-β-d-galactoside (IPTG), and the recombinant proteins with high purity were obtained. Western blotting showed that the recombinant proteins could be recognized by the corresponding antibodies. The target proteins were identified as multimers with α helixes by native-PAGE and circular dichroism. In vitro and in vivo tumor uptake experiments demonstrated that fusion ferritin was taken up by tumor cells and tumor tissue. This study successfully expressed, purified, and identified fusion ferritin, and verified its tumor uptake in vitro and in vivo, which laid a foundation for the application of ferritin in biomedicine.