TAT介导的金属硫蛋白的穿膜效应及对细胞氧化损伤的修复作用

Cell-penetrating Effects and Repair of Cell Oxidative Damage of TAT-metallothionein Fusion Proteins

人类免疫缺陷病毒反式激活因子(human immunodeficiency virus transactivator,TAT)蛋白质转导肽是HIV-1编码的反式转录激活因子,富含碱性氨基酸序列,能够高效介导多种外源生物大分子通过多种膜性结构,例如细胞质膜和血脑屏障等。金属硫蛋白(metallothionein,MT)是一类低分子量、富含半胱氨酸的蛋白质,在维持生物体内金属含量动态平衡、重金属解毒以及防御氧化应激中均发挥重要作用。本研究通过基因重组技术制备由TAT介导的能跨膜进入细胞的重组融合蛋白质TAT-MT,使其进入细胞,高效发挥抗氧化损伤效应。通过体外抗氧化实验测定TAT-MT的体外羟自由基清除率及总抗氧化能力;免疫荧光技术检测其穿膜活性;MTT法研究其对H2O2诱导的293T细胞氧化损伤的修复作用。结果显示,通过长引物PCR技术在目的基因5′-端加入TAT序列,成功构建原核表达载体pET28a-TAT-MT;通过TAT标签的引入显著增加了MT的可溶性表达;进一步通过表达条件的优化成功获得了TAT-MT的高效可溶性表达;采用亲和层析方法对重组蛋白质进行了分离纯化;免疫荧光检测显示,重组蛋白质可以高效进入细胞;体外抗氧化结果显示,当TAT-MT浓度为100μmol/L时,羟自由基清除率达到94.87%±5.18%,说明TAT-MT具有较强的清除羟自由基的能力;MTT结果表明,重组蛋白质对H2O2诱导的293T细胞氧化损伤具有显著的(P<0.05)修复作用。本研究为MT的规模化制备及进一步开发其在生物医药、食品保健及化妆品领域的应用奠定了基础。

The human Immunodeficiency Virus Transactivator(TAT)protein transduction peptide is a trans-transcription activator encoded by HIV-1.It is rich in basic amino acids,and capable of efficiently mediating the passage of exogenous macromolecules through a variety of membrane structures,such as the cytoplasmic membrane and the blood-brain barrier.Metallothionein(MT)is a protein with low molecular weights and rich cysteine contents.It plays important roles in maintaining the dynamic balance of metal contents in the body,in the detoxification of heavy metals and in defense against oxidative stress.Based on the full-length MT cDNA previously cloned from Sinopotamon henanense,we aim to prepare a TAT-mediated recombinant fusion protein that can cross the membrane and enter the cell by means of genetic engineering.The hydroxyl radical scavenging rate and total antioxidant capacity of TAT-MT were measured in vitro.An immunofluorescence technique was used to detect the transmembrane activity.An MTT assay was used to study the repair effect of H2O2-induced oxidative damage of 293 T cells.The results showed that pET28 a-TAT-MT was successfully constructed by adding the TAT sequence into the target gene using a long primer PCR.The high soluble expression of the recombinant protein TAT-MT in Escherichia coli was obtained by optimizing expression conditions.The recombinant protein was isolated and purified by affinity chromatography.Immunofluorescence showed that the recombinant protein could enter the cells.In vitro antioxidant experiments showed that TAT-MT had high hydroxyl radical scavenging capacity and total antioxidant capacity.The MTT assay showed that the recombinant protein had a significant repair effect on H2O2-induced oxidative damage of 293 T cells.This study lays the foundation for the further development and application of MT.