Transient expression of CCL21 chemokine in chickpea via agroinfiltration

Document Type : Original Articles

Authors

• Maria Beihaghi ¹
• Abdol reza Bagheri ^{2, 3}
• Hasan Marashi ²
• Mojtaba Sankian ⁴
• Afsaneh Sadat Farsad ⁵

¹ Department of Biology, Kavian Institute of Higher Education, Mashhad, Iran

² Department of Biotechnology, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

³ Research Center for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

⁴ BuAli Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran

⁵ College of Agriculture, University of Birjand, Birjand, Iran

10.22067/ijpr.v10i1.60228

Abstract

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.

Keywords

• Agro-infiltration
• C-C chemokine ligand 21 (CCL21)
• Chickpea
• Transient gene expression

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References

1. Bendahmane, A., Querci, M., Kanyuka, K., and Baulcombe, D.C. 2000. Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. The Plant Journal 21(1): 73-81.
2. Boehm, R. 2007. Bioproduction of therapeutic proteins in the 21^st century and the role of plants and plant cells as production platforms. Annals of the New York Academy of Sciences 1102(1): 121-134.
3. Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-54.
4. Ding, Y., Shimada, Y., Maeda, M., Kawabe, A., Kaganoi, J., Komoto, I., and Imamura, M. 2003. Association of CC chemokine receptor 7 with lymph node metastasis of esophageal squamous cell carcinoma. Clinical Cancer Research 9(9): 3406-3412.
5. Farsad, A.S., Malekzadeh-Shafaroudi, S., Moshtaghi, N., Fotouhi, F., and Zibaee, S. 2016. Transient expression of HA1 antigen of avian influenza virus (H5N1) in alfalfa, soybean and lettuce leaves by agroinfiltration. Kerman Journal of Biotechnology 8 (1): 61-80.
6. Fischer, R., Stoger, E., Schillberg, S., Christou, P., and Twyman, R. 2004. Plant based production of Biopharmaceuticals. Current Opinion in Plant Biology 7: 152-158.
7. Floss, D.M., Falkenburg, D., and Conrad, U. 2007. Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview. Transgenic Research 16(3): 315-332.
8. Gidding, G., Allison, G., Brooks, D., and Carter, A. 2000. Transgenic plants as factories for b Natural Biotechnology 18: 1151-1155.
9. Gollmer, K., Asperti-Boursin, F., Tanaka, Y., Okkenhaug, K., Vanhaesebroeck, B., Peterson, J.R., Fukui, Y., Donnadieu, E., and Stein, J.V. 2009. CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway. Blood 114: 580-
10. Habibi-Pirkoohi, M., Malekzadeh-Shafaroudi, S., Marashi, H., Moshtaghi, N., Nassiri, M.R., and Zibaee, S. 2014. Transient expression of foot and mouth disease virus (FMDV) coat protein in tobacco (Nicotiana tabacom) via a Iranian Journal of Biotechnology 12: 28-35.
11. Hashemi, H., Jourabchi, E., and Khodabandeh, M. 2005. Transient expression of human growth hormone in potato (Solanum tuberosum), tobacco (Nicotiana tobacum) and lettuce (Lactuca sativa) leaves by agroinfiltration. Iranian Journal of Biotechnology 3(2): 109.
12. Hefferon, K., Grealy, M., and Mutrie, N. 2010. Transforming from Cocoon to Butterfly: The potential role of the body in the process of posttraumatic g Journal of Humanistic Psychology 50(2): 224-247.
13. Hobbs, S.L.A., Jackson, J.A., and Mahon, J.D. 1989. Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens. Plant Cell Reports 8: 274-277.
14. Johnson, N., Bryant, A., Miles, T., Hogberg, T., and Cornes, P. 2011. Adjuvant Chemotherapy for Endometrial Cancer after H The Cochrane Database of Systematic Reviews.
15. Kim, T.G., and Yang, M.S. 2010. Current trends in edible vaccine development using transgenic p Biotechnology and Bioprocess Engineering 15: 61-65.
16. Koprowski, H., and Yusibov, V. 2001. The green revolution: plants as heterologous expression vectors. Vaccine 19(17): 2735-2741.
17. Kozak, M. 1989. The scanning model for translation: an update. The Journal of Cell Biology 108(2): 229-241.
18. Leckie, B.M., and Stewart, C.N. 2011. Agroinfiltration as a technique for rapid assays for evaluating candidate insect resistance transgenes in plants. Plant Cell Reports 30(3): 325-334.
19. Livak, K.J., and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt Methods 25: 402-408.
20. Lollini, P.L., Cavallo, F., Nanni, P., and Forni, G. 2006. Vaccines for tumour prevention. Nature Reviews Cancer 6(3): 204-16.
21. Lu, L.L., Chen, X.H., Zhang, G., Liu, Z.C., Wu, N., Wang, H., Qi, Y.F., Wang, H.S., Cai, S.H., and Du, J. 2016. CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3β/Snail Signals. Oxidative Medicine and Cellular Longevity.
22. Mantovani, A., Bonecchi, R., and Locati, M. 2006. Tuning inflammation and immunity by chemokine sequestration: decoys and more. Nature Reviews Immunology 6(12): 907-918.
23. Mehrotra M, Sanyal, I., Amla, D.V. 2011. High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum) and regeneration of insect-resistant transgenic plants. Plant Cell Reports 30: 1603-1616.
24. Muller, A., Homey, B., Soto, H., Ge, N.F., Catron, D., Buchanan, M.E., and Wagner, S.N. 2001. Involvement of chemokine receptors in breast cancer metastasis. Nature 410(6824): 50-56.
25. Nie, L., Wu, G., and Zhang, W. 2006. Correlation between mRNA and protein abundance in Desulfovibrio vulgaris: a multiple regression to identify sources of variations. Biochemical and Biophysical Research Communications 339: 603-610.
26. Obembe, O.O., Popoola, J.O., Leelavathi, S., and Reddy, S.V. 2011. Advances in plant molecular farming. Biotechnology Advances 29: 210-222.
27. Sain, M., and Sharma, V. 2013. Catharanthus roseus (An anti-cancerous drug yielding plant)- A review of potential Therapeutic p International Journal of Pure & Applied Bioscience 1(6): 139-142.
28. Shivani I., Hari, S., and Susan, E., 2010. Agrobacteriium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors. Physiol Molecular Biology Plants 16(3): 273-284.
29. Shoji, Y., Bi, H., Musiychuk, K., Rhee, A., Horsey, A., and Roy, G. 2009. Plant-derived hemagglutinin protects ferrets against challenge infection with the A/Indonesia/05/05 strain of avian influenza. Vaccine 27: 1087-
30. Simmons, C.W., VanderGheynst, J.S., and Upadhyaya, S.K. 2009. A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnology and Bioengineering 102(3): 965-970.
31. Wandeltt, C., Rafiqul, M., Khan, S.C., Harmut, E.S., Spencer, D., and Higgins, T.J.V. 1992. Vicilin with carboxyterminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant Journal 2: 181-192.
32. Wang, X.Q. 2004. 5-Untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation. Nucleic Acids Research 32: 1382-1391.
33. Wang, A., and Ma, S. 2012. Molecular Farming in Plants: Recent Advances and Future Prospects. Springer 183-198.
34. Wang, J., Seethala, R.R., Zhang, Q., Gooding, W., Van Waes, C., Hasegawa, H., and Ferris, R.L. 2008. Autocrine and paracrine chemokine receptor 7 activation in head and neck cancer: implications for therapy. Journal of the National Cancer Institute 100 (7): 502-512.
35. Watson, M., Benard, V., Thomas, C., Brayboy, A., Paisano, R., and Becker, T. 2014. Cervical cancer incidence and mortality among American Indian and Alaska Native women, 1999-2009. American Journal of Public Health 104 (3): S415-22.
36. Ware, M. 2016. Chickpeas: Health Benefits, Nutritional Information. Available at Web site http://www.medicalnewstoday.com.
37. Wroblewski, T., Tomczak, A., and Michelmore, R. 2005. Optimization of Agrobacterium‐mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnology Journal 3(2): 259-273.
38. Yajun, F., Wei, L., Xiaojuan, Z., and Xingzhi, W. Transient expression of acidic fibroblast growth factor in pea (Pisum sativum L.) plants. African Journal of Biotechnology 11(22): 6060-6063.