College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.
Department of Biology, Kavian Institute of Higher Education, Mashhad, Iran.
PLoS One. 2022 Aug 29;17(8):e0261101. doi: 10.1371/journal.pone.0261101. eCollection 2022.
CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7 cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.
CCL21 在抗肿瘤免疫活性中具有重要作用。IL1β 的表位具有佐剂活性，而不会引起炎症反应。CCR7 及其配体在免疫平衡中起着至关重要的作用；具体而言，在 T 淋巴细胞和抗原呈递细胞（如树突状细胞）向淋巴结的运输中起着至关重要的作用。本研究旨在产生 CCL21 和 IL1β 的表位作为重组蛋白，并表征其体外抗肿瘤和免疫原性活性。从人基因中设计并合成了经过密码子优化的 ccl21/IL1β 基因。通过计算机分析研究了 CCL21/IL1β 蛋白和 CCR7 受体的稳定性和结合亲和力。将构建体引入到 N. tabacum 中以产生这种重组蛋白，并检查 CCL21/IL1β 的结构和功能。从转基因叶片中纯化的蛋白在 SDS PAGE 和 western blotting 分析中产生了强烈的信号。FTIR 测量和 MALDI-TOF/TOF 质谱分析表明，ccl21/IL-1β 在烟草植物中正确表达。使用划痕愈合方法研究了纯化的 CCL21/IL1β 在刺激 MCF7 癌细胞系增殖和迁移中的潜在活性。结果表明，在 CCL21/IL1β 的存在下，癌细胞的存活率和转移率降低，并且 CCL21 在 MCF7 细胞上的 IC50 小于非重组蛋白。对 PBMCsCCR7+的琼脂糖测定表明，CCL21/IL1β 具有生物活性，并且趋化性（CCL21）和趋化性（FBS）运动之间存在明显差异。总的来说，这些结果表明 CCL21/IL1β 可以被认为是未来体内和临床测试中的有效佐剂。
