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Transfer and expression of exendin-4, stimulant of insulin secretion, fused to cholera toxin B subunit (CTB) in lettuce
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Bahman Bahramnejad *    , Ebrahim Moradi , Mohammad Naji , Hemn Salehi |
| University of Kurdistan , b.bahramnejad@uok.ac.ir |
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Abstract: (124 Views) |
Due to the ability to produce recombinant proteins at low cost, high volume of biomass and high safety, plants can be considered as a suitable alternative for the common systems of producing recombinant pharmaceutical proteins. Glucan-like peptide (GLP-1) plays a role in inducing insulin secretion in the body. Exendin-4 (EX4) is a protein similar to GLP-1, with the difference that it has a longer half-life and thus a greater effect on inducing insulin secretion. Fusing the EX4 to the non-toxic cholera subunit (CTB) increases its efficiency through absorption in the digestive system. In this research, vector pBI121 containing CTB-EX4 fragment was introduced into lettuce nucleus by Agrobacterium tumefaciens. 20.25% of the inoculated explants were successfully transformed and regenerated and after confirmation in the PCR test, first generation transgenic seeds (T0) were prepared from the plants obtained from these micro-samples. In order to create second generation plants (T1), a total of 24 T0 seeds were cultivated, 42% of the cultivated seeds grew fully and set to seed. Using specific primers in the PCR reaction, transgenesis and the presence of CTB-EX4 gene in T1 plants were investigated. The semi-quantitative and quantitative expression of CTB-EX4 gene was investigated at the mRNA level using RT-PCR and Real time PCR tests and at the protein level using specific antibodies and ELISA and Western Blot tests. Finally, the number of transferred copies to T1 plants was estimated using the Real time PCR by the standard curve as well as the relative method, compared to the internal control gene Actin. The results showed that 60% of the second generation lines (T1) were transgenic and the CTB-EX4 gene was expressed at the level of mRNA and protein. The results showed that the amount of expression was different between studied lines. The highest amount of expression at the protein and mRNA levels was observed in line of 105. The highest number of transferred copies was observed in line 101 and the least number of transferred copies was 1 copy in lines 104 and 105. The results showed that the process of transferring the CTB-EX4 gene to the lettuce plant was successful and the lettuce plant was able to express the CTB-EX4 gene at the RNA and protein levels. Considering the subsequent processes, including the purification and evaluation of the biological activity of the produced protein, plants can be used as a suitable bioreactor for the production of recombinant proteins.
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| Keywords: Agrobacterium tumefaciens, Exendin, gene expression, pBI121 vector, Recombinant protein. |
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Type of Study: Research |
Subject:
Plant
Received: 2023/07/2 | Accepted: 2023/08/29 | Published: 2024/03/14
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