In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1 beta 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 IL1 beta as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1 beta gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1 beta 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/IL1 beta 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-1 beta was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1 beta 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/IL1 beta, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1 beta has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1 beta could be considered an effective adjuvant in future in vivo and clinical tests.

Automated summary

This study aimed to produce epitopes of CCL21 and IL1 beta as a recombinant protein and evaluate its in vitro anti-tumor and immunogenic activity. The results demonstrated that CCL21/IL1 beta could decrease the survival rate and metastasization of cancer cells, and it has biological activity as a potential effective adjuvant in future in vivo and clinical tests.