Abstract：
Most plant and bacterial toxins are highly immunogenic with non-specific toxic effects. Human ribonucleases are thought to provide a promising basis for reducing the toxic agent\'s immunogenic properties, which are candidates for cancer therapy. In the cell, the ribonuclease inhibitor (RI) protein binds to the ribonuclease enzyme and forms a tight complex. This study aimed to engineer and provide a gene construct encoding an improved version of Human Pancreatic RNase 1 (HP-RNase 1) to reduce connection to RI and modulate the immunogenic effects of immunotoxins. To further characterize the interaction complex of HP-RNase 1 and RI, we established various in silico and in vitro approaches. These methods allowed us to specifically monitor interactions within native and engineered HP-RNase 1/RI complexes. In silico research involved molecular dynamics (MD) simulations of native and mutant HP-RNase 1 in their free form and when bound to RI. For HP-RNase 1 engineering, we designed five mutations (K8A/N72A/N89A/R92D/E112/A) based on literature studies, as this combination proved effective for the intended investigation. Then, the cDNA encoding HP-RNase 1 was generated by RT-PCR from blood and cloned into the pSYN2 expression vector. Consequently, wild-type and the engineered HP-RNase 1 were over-expressed in E. coli TG1 and purified using an IMAC column directed against a poly-his tag. The protein products were detected by SDS-PAGE and Western blot analysis. HP-RNase 1 catalytic activity, in the presence of various concentrations of RI, demonstrated that the mutated version of the protein is able to escape the ribonuclease inhibitor and target the RNA substrate 2.5 folds more than that of the wild type. From these data, we tend to suggest the engineered recombinant HP-RNase 1 potentially as a new immunotherapeutic agent for application in human cancer therapy.
摘要：
大多数植物和细菌毒素具有高度免疫原性,具有非特异性毒性作用。人核糖核酸酶被认为为降低毒性剂的免疫原性提供了有希望的基础，是癌症治疗的候选人。在牢房里，核糖核酸酶抑制剂(RI)蛋白与核糖核酸酶结合并形成紧密复合物。这项研究旨在设计并提供编码人胰腺RNase1（HP-RNase1）的改良版本的基因构建体，以减少与RI的连接并调节免疫毒素的免疫原性作用。为了进一步表征HP-RNase1和RI的相互作用复合物，我们建立了各种计算机和体外方法。这些方法使我们能够特异性监测天然和工程化HP-RNase1/RI复合物内的相互作用。计算机研究涉及游离形式和与RI结合时的天然和突变体HP-RNase1的分子动力学（MD）模拟。对于HP-RNase1工程，我们在文献研究的基础上设计了5种突变(K8A/N72A/N89A/R92D/E112/A),因为这种组合被证明对预期的调查是有效的。然后，通过RT-PCR从血液中产生编码HP-RNase1的cDNA，并将其克隆到pSYN2表达载体中。因此,野生型和工程化的HP-RNase1在大肠杆菌TG1中过表达,并使用针对polyhis标签的IMAC柱纯化。通过SDS-PAGE和Western印迹分析检测蛋白质产物。HP-RNase1催化活性,在各种浓度的RI存在下，表明突变版本的蛋白质能够逃脱核糖核酸酶抑制剂，并靶向RNA底物比野生型多2.5倍。从这些数据来看,我们倾向于建议工程重组HP-RNase1可能作为一种新的免疫治疗剂应用于人类癌症治疗。
