Expression of ccl21 protein in tobacco (nicotiana tabacum) and characterization of its anti-tumor activity in ex-vivo

Chemokines and their receptors play essential roles in leukocyte recruitment, tumor cell growth and metastasis. Chemokine (C-C motif) ligand 21 (CCL21) is a small cytokine belonging to the CC chemokine family. CCL21 protein has a fundamental role in anti-tumor immune activity. Hence, it is considered as a potential vaccine candidate. Application of plants as bioreactors for production ofrecombinant proteins because of safe and cost effective has emerged as a molecular farming over the past two decades. The aim of this study was to obtain the possibility of producing recombinant CCL21in Nicotiana tabacum via stable transformation by agrobacterium and perform quantitative analysis and functionality of CCL21by various methods. We designed and synthesized a codon-optimized gene(ccl21/IL1β ) from the CCL21 and part of interleukin 1β by bioinformatics analysis. CCl21 construct was transformed to tobacco leaves. Then, resulted plants were analyzed by Real-time PCR, protein dotblot, enzyme-linked immunosorbent assay (ELISA), Western blotting and FTIR spectroscopy. Results confirmed presence of recombinant protein in transgenic lines. Also, functionality of the produced CCL21 was analyzed on human breast cancer cell line MCF7, and peripheral blood mononuclear cell (PBMC) by cytotoxicity, scratch and migration assays. The cytotoxic of CCL21 plant recombinant protein was investigated on MCF7 by MTT assay. We developed an agarose-based chemotaxis assay, which was tested in PBMC, to follow T cells moving toward chemoattractant. Also, scratch assay wasused to investigate the role of this protein in anti-metastatic function. Our results suggested that CCL21 plant recombinant protein can be considered as an effective anticancer agent for future in vivoand clinical experiments. Key words: CCL21, FTIR, Scratch assay, Chemotaxis assay, MTT assay

کلیدواژه ها:

Breast Cancer ، Lung Cancer ، Gene and Cancer ، Cancer Genetics ، Cancer Treatment and Management