بررسی بیان موقت پروتئین نوترکیبCCL21 انسانی در گیاه نخود به روش آگرواینفیلتراسیون مقاله
پژوهش های حبوبات ایران بهار و تابستان 1398 - شماره 19 علمی-پژوهشی (وزارت علوم)/ISC (12 صفحه - از 91 تا 102)
کموکاین CCL21 از جمله کموکاینهایی است که خاصیت آنتیتوموری دارد و در ایمونوتراپی بر پایه سیتوکنینها بر علیه سلولهای سرطانی موثر میباشد. با توجه به اهمیت فناوری جدید تولید پروتئینهای نوترکیب در گیاهان، تولید پروتئین CCL21 نیز در گیاه نخود که دارای خواص آنتیتوموری و ترکیبات معدنی و شیمیایی مناسب است، مورد توجه قرار گرفته است. در مطالعه حاضر از بیان موقت پروتئین CCL21 به روش آگرواینفیلتراسیون، برای تولید این پروتئین نوترکیب در گیاه نخود استفاده شده است. 72ساعت بعد از آگرواینفیلتراسیون، نتایج آزمونهای RT-PCR، Real time PCR، داتبلات، وسترنبلات و الایزا نشان داد که سازه ژنی طراحی شده در برگهای نخود با میزان بالایی در سطح رونویسی و ترجمه بیان شده است. بنابراین با استفاده از روش آگرواینفیلتراسیون و انتقال سازه بیانی CCL21، نتایج نشان داد که نخود میتواند گیاه مناسبی جهت تولید پروتئینهای نوترکیب باشد.
Introduction Chemokine (C-C motif) ligand 21 (CCL21) has anti-tumor efficacy and used for immunotherapy based on cytokinins against cancer cells. Immune potentiating of CCL21 chemokine via DC and stromal cell-based approaches for effective recruitment and activation of APC and T cells for promotion of antitumor activity in lung cancer was evaluated. Plants are now gaining widespread acceptance as a general platform and as bioreactors for the large scale production of recombinant proteins. However, the technique suffers from major drawbacks such as low expression level and long time required for the production of the recombinant protein in plant tissues. The goal of the present study was to investigate the possibility of expressing the CCL21 protein in Cicer arietinum via agroinfiltration. In agroinfiltration, the suspension of Agrobacterium tumefaciens harboring the gene(s) of interest is infiltrated into plant leaves using a needle-free syringe. This technique has been carried out in a variety of plants with different experimental purposes and it was simple, cost effective and rapid procedure. Chickpea contain several minerals and phytochemicals components for human health, for example it has selenium, that plays essential roles in liver enzyme function, and helps detoxify some cancer-causing compounds in the body, Also selenium prevents inflammation and also decreases tumor growth rates. So cheakpea known as a valuable plant for the production of recombinant proteins chickpeas also contains folate, which plays a role in DNA synthesis and repair, thus preventing the formation of cancer cells from mutations in the DNA. Also it has saponins, which are phytochemicals that prevent cancer cells from multiplying and spreading throughout the body. High-fiber intakes from fruits and vegetables like chickpeas are associated with a lowered risk of colorectal cancer. Therefore, in this study, ccl21 gene of human was synthesized and transiently expressed in Cicer arietinum. Materials & Methods To examine the possibility of expressing the CCL21 protein in Cicer arietinum, a cDNA fragment encoding the ccl21 gene was synthesized de novo, modified with a Kozak sequence, a C-terminal hexa-histidine (6His) tag, and an endoplasmic retention signal (SEKDEL). The construct was subcloned into vector pBI121 and Agrobacterium colonies were verificated by PCR test. The resulting ccl21 plasmid was agro-infiltrated into Cicer arietinum. The relative gene expression of recombinant plant-produced ccl21 was measured by semi quantitatve RT-PCR and quantitative real-time PCR. Guided by the gene expression profile, CCL21 protein was extracted after 72 hours. A recombinant CCL21 protein was immunogenically detected by conjugated polyhistidine antibody in western blot, dot blot and ELISA assay. Results & Discussion The results of PCR assay confirmed presence of the synthetic construct in Agrobacterium clones. Also PCR test showed that, this construct was integrated in infiltrated leaves but the gene of interest is not integrated in the nuclear genome of plant cell. So, the transgene in plant tissue is higher than pure plasmid as control. RT- PCR and Real time PCR assay was also conducted to evaluate the expression of gene. Dot blot assay showed that, the protein sample obtained from transformed leaves generated a strong signal which is comparable to that of positive control, whereas wild type plant protein was not detectable. Also enzyme-linked immunosorbent assay (ELISA) and western blot showed that gene construt of ccl21 was expressed highly in transcription and translation level. Migration size of protein was detected at 15.5 KD by Western blotting. ELISA results showed that the CCL21 was expressed in the transfected leaves in high level. Conclusion This paper is the first research about the transient expression of the chickpea-made CCL21 protein where a synthetic sequence was used for its expression. Here, the efficacy of agroinfiltration for expression of ccl21 gene in chickpea was illustrated. Agroinfiltration expedites the process of recombinant protein expression in plant tissues. Therefore, using agro-infiltration system in chickpea plant was the appropriate strategy for ccl21 gene production in the assayed plants. Therefore, this method makes it possible to evaluate efficacy of a potential recombinant vaccine in a short time.
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