ﻃﺮاﺣﯽ ﺑﯿﻮاﻧﻔﻮرﻣﺎﺗﯿﮑﯽ و ﻫﻤﺴﺎﻧﻪﺳﺎزي ژن rT1 ﭼﺎي ﺗﺮش ﺑﺮاي ﺳﺎﺧﺖ وﮐﺘﻮر ﻧﻮﺗﺮﮐﯿﺐ pBI121-rT1 ﺑﻪ ﻣﻨﻈﻮر ﺑﯿﺎن در ﮔﯿﺎﻫﺎن

Bioinformatics design and cloning of Hibiscus sabdariffa rT1 gene for construction of pBI121-rT1 recombinant vector in order to expression in plants

ﺑﯿﻤﺎريﻫﺎي ﺗﻨﻔﺴﯽ و اﻟﺘﻬﺎب رﯾﻮي از ﺷﺎﯾﻊﺗﺮﯾﻦ ﺑﯿﻤﺎريﻫﺎﯾﯽ اﺳﺖ ﮐﻪ ﺑﯿﺶ از 50 درﺻﺪ ﻣﺮدم ﺟﻬﺎن را درﮔﯿﺮ ﻣﯽﮐﻨﺪ. ﺗﺎﮐﻨﻮن داروﻫﺎي ﻣﺨﺘﻠﻔﯽ در زﻣﯿﻨﻪ ﭘﯿﺸﮕﯿﺮي و درﻣﺎن اﯾﻦ ﺑﯿﻤﺎريﻫﺎ ﺗﻮﺳﻌﻪ ﯾﺎﻓﺘﻪ اﺳﺖ. ﭘﭙﺘﯿﺪ rT1، ﻣﺘﻌﻠﻖ ﺑﻪ ﮔﯿﺎه داروﯾﯽ ﭼﺎي ﺗﺮش ﺑﺎ ﻃﻮل 27 اﺳﯿﺪآﻣﯿﻨﻪ داراي ﺧﺎﺻﯿﺖ ﻣﻬﺎرﮐﻨﻨﺪﮔﯽ ﻧﻮﺗﺮوﻓﯿﻞ اﻻﺳﺘﺎز اﻧﺴﺎﻧﯽ و ﭘﺘﺎﻧﺴﯿﻞ ﺑﺎﻟﻘﻮه داروﯾﯽ در زﻣﯿﻨﻪ ﭘﯿﺸﮕﯿﺮي و درﻣﺎن ﺑﯿﻤﺎري-ﻫﺎي اﻟﺘﻬﺎﺑﯽ رﯾﻪ اﺳﺖ. ﺑﺎﺗﻮﺟﻪ ﺑﻪ ارزش اﻗﺘﺼﺎدي ﺑﺎﻻي ﺗﺮﮐﯿﺒﺎت ﺑﺎ ﺧﺎﺻﯿﺖ ﻣﻬﺎرﮐﻨﻨﺪﮔﯽ ﻧﻮﺗﺮوﻓﯿﻞ اﻻﺳﺘﺎز، در اﯾﻦ ﭘﮋوﻫﺶ ﺑﺮاي اوﻟﯿﻦ ﺑﺎر ﺑﺎ ﻫﺪف ﺑﯿﺎن اﯾﻦ ﭘﭙﺘﯿﺪ در ﮔﯿﺎﻫﺎن، ﻫﻤﺴﺎﻧﻪﺳﺎزي و ﺳﺎﺧﺖ وﮐﺘﻮر ﺑﯿﺎﻧﯽ ﻧﻮﺗﺮﮐﯿﺐ pBI121-rT1 اﻧﺠﺎم ﺷﺪ. ﺑﺎﺗﻮﺟﻪ ﺑﻪ ﻧﺒﻮدن ﺗﻮاﻟﯽ ژﻧﯽ اﯾﻦ ﭘﭙﺘﯿﺪ در ﭘﺎﯾﮕﺎه-ﻫﺎي داده، اﺑﺘﺪا ﺗﻮاﻟﯽ ژﻧﯽ ﻣﻨﺎﺳﺐ آن ﺑﺮاﺳﺎس ﺗﺮﺟﯿﺢ ﮐﺪوﻧﯽ ﮔﯿﺎه ﺗﻮﺗﻮن، وﮐﺘﻮرﻫﺎي ﺑﮑﺎر ﺑﺮده ﺷﺪه در ﭘﮋوﻫﺶ، ﺳﺎﺧﺘﺎر و ﭘﺎﯾﺪاري روﻧﻮﺷﺖ ﺣﺎﺻﻞ از آن و ﺧﺼﻮﺻﯿﺎت ﭘﺮوﺗﺌﯿﻦ ﺗﻮﻟﯿﺪ ﺷﺪه از آن ﻃﺮاﺣﯽ و ﺳﻨﺘﺰ ﺷﺪ. ﺷﺎﺧﺺ ﺳﺎزﮔﺎري ﮐﺪوﻧﯽ ﺑﺮاي ﺗﻮاﻟﯽ ﻃﺮاﺣﯽ ﺷﺪه ﺑﺮاﺑﺮ ﺑﺎ 0/92 و ﻓﺮاواﻧﯽ GC 42/32 درﺻﺪ ﻣﺤﺎﺳﺒﻪ ﺷﺪ. ﭘﺎﯾﺪاري ﭘﭙﺘﯿﺪ ﻃﺮاﺣﯽ ﺷﺪه در ﺳﻠﻮلﻫﺎي اﻧﺴﺎﻧﯽ ﻧﺴﺒﺖ ﺑﻪ ﭘﭙﺘﯿﺪ اﺻﻠﯽ 25 ﺑﺮاﺑﺮ اﻓﺰاﯾﺶ ﯾﺎﻓﺖ و ﺷﺎﺧﺺ ﻧﺎﭘﺎﯾﺪاري ﺑﺮاي ﭘﭙﺘﯿﺪ ﻃﺮاﺣﯽ ﺷﺪه ﺑﺮاﺑﺮ 20/90 ﻣﺤﺎﺳﺒﻪ ﺷﺪ. ﺑﻪ ﻣﻨﻈﻮر ﻫﻤﺴﺎﻧﻪﺳﺎزي، ﺗﻮاﻟﯽ ﺳﻨﺘﺰ ﺷﺪه ﺑﻪ وﺳﯿﻠﻪ واﮐﻨﺶ PCR ﺗﮑﺜﯿﺮ و در وﮐﺘﻮر pMCS5 ﻫﻤﺴﺎﻧﻪﺳﺎزي ﺷﺪ. ﺳﭙﺲ ﺑﺎ ﻫﺪف ﺑﯿﺎن اﯾﻦ ﭘﭙﺘﯿﺪ در ﮔﯿﺎﻫﺎن، زﯾﺮﻫﻤﺴﺎﻧﻪﺳﺎزي ﺗﻮاﻟﯽ ﻃﺮاﺣﯽ ﺷﺪه، در وﮐﺘﻮر ﺑﯿﺎﻧﯽ pBI121 ﺗﺤﺖ ﭘﺮوﻣﻮﺗﺮ CaMV 35S اﻧﺠﺎم ﺷﺪ.

Respiratory diseases and pneumonia are among the most common diseases affecting more than 50% of the world's population. Various drugs have been developed to prevent and treat these diseases. rT1 peptide is a plant peptide isolated from the medicinal plant of Hibiscus sabdariffa. This 27-amino-acid peptide with inhibitory properties of human neutrophil elastase has been shown to have drug potential in the prevention and treatment of inflammatory lung diseases. Due to the high economic value of the compounds with neutrophil elastase inhibitory properties, in this study, for the first time, recombinant expression vector pBI121-rT1 was constructed with the aim of expressing rT1 peptide in plants. Because of the lack of rT1 gene sequence in databases, first, its appropriate gene sequence was designed and synthesized based on the Nicotiana tubaccum codon usage, the vectors used in this research, the structure and stability of its transcript and characteristics of the produced protein. For the designed sequence, the Codon Adaptation Index (CAI) and the GC frequency were 0.92 and 42.32%, respectively. In comparison to the main peptide, the stability of the designed peptide in human cells increased 25 times and its instability index calculated to be 20.90. Following amplification of the synthesized sequence by PCR reaction, it was cloned into pMCS5 vector. In order to express in plant-based systems, sub-cloning was performed in the expression vector pBI121 under the CaMV 35S promoter.