پديد آورندگان :
ﻗﻮﯾﻤﯽ، ارد داﻧﺸﮕﺎه ﺗﻬﺮان - داﻧﺸﮑﺪه ﻋﻠﻮم و ﻓﻨﻮن ﻧﻮﯾﻦ - ﮔﺮوه ﻣﻬﻨﺪﺳﯽ ﻋﻠﻮم زﯾﺴﺘﯽ، ﺗﻬﺮان، اﯾﺮان , ﺣﺎﺟﯽ ﺣﺴﻦ، زﻫﺮا داﻧﺸﮕﺎه ﺗﻬﺮان - داﻧﺸﮑﺪه ﻋﻠﻮم و ﻓﻨﻮن ﻧﻮﯾﻦ - ﮔﺮوه ﻣﻬﻨﺪﺳﯽ ﻋﻠﻮم زﯾﺴﺘﯽ، ﺗﻬﺮان، اﯾﺮان , ارﻣﻐﺎن، ﻓﺎﻃﻤﻪ داﻧﺸﮕﺎه ﺗﻬﺮان - داﻧﺸﮑﺪه ﻋﻠﻮم و ﻓﻨﻮن ﻧﻮﯾﻦ - ﮔﺮوه ﻣﻬﻨﺪﺳﯽ ﻋﻠﻮم زﯾﺴﺘﯽ، ﺗﻬﺮان، اﯾﺮان
كليدواژه :
اﺷﺮﺷﯿﺎﮐﻠﯽ , ﭘﺮوﺗﺌﯿﻦ ﻧﻮﺗﺮﮐﯿﺐ , ﭘﺮوﺗﺌﯿﻦ ﻣﺤﻠﻮل , اﮐﺘﯿﻮﯾﻦ A
چكيده فارسي :
اﮐﺘﯿﻮﯾﻦ A ﯾﮑﯽ از اﻋﻀﺎي ﺧﺎﻧﻮاده ﻓﺎﮐﺘﻮر رﺷﺪ ﺗﻐﯿﯿﺮدﻫﻨﺪه ﺑﺘﺎ )TGF-β( اﺳﺖ ﮐﻪ ﻧﻘﺶ ﻣﻬﻤﯽ در ﻓﺮاﯾﻨﺪﻫﺎي ﻓﯿﺰﯾﻮﻟﻮژﯾﮑﯽ ﻣﺘﻌﺪد ﻫﻤﺎﻧﻨﺪ ﺗﻤﺎﯾﺰ ﺳﻠﻮﻟﯽ، ﺗﺮﻣﯿﻢ ﺑـﺎﻓﺘﯽ، رگ زاﯾـﯽ، ﺗﻤـﺎﯾﺰ ﺳـﻠﻮل ﻫـﺎي ﺑﻨﯿﺎدي، ﭼﺴﺒﻨﺪﮔﯽ ﺳﻠﻮﻟﯽ و آﭘﻮﭘﺘﻮز دارد. ﺑﻨﺎﺑﺮاﯾﻦ ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﮐﺎرﺑﺮدﻫﺎي ﺑﺎﻟﯿﻨﯽ ﻣﺘﻌﺪد اﯾﻦ ﭘﺮوﺗﺌﯿﻦ، ﺗﻮﻟﯿﺪ ﻧﻮﺗﺮﮐﯿﺐ آن ﺳﻮدﻣﻨﺪ ﻣﯽﺑﺎﺷﺪ. از آﻧﺠﺎﯾﯽﮐﻪ اﺷﺮﺷﯿﺎﮐﻠﯽﯾﮑﯽ از ﻣﺤﺒﻮبﺗﺮﯾﻦ ﻣﯿﺰﺑﺎنﻫﺎ ﺑﺮاي ﺗﻮﻟﯿﺪ ﭘﺮوﺗﺌﯿﻦﻫﺎي ﻧﻮﺗﺮﮐﯿﺐ اﺳﺖ، در اﯾﻦ ﺗﺤﻘﯿﻖ از ﺑﯿﺎن ﺳﯿﺘﻮﭘﻼﺳﻤﯽ در اﯾﻦ ﺳﻮﯾﻪ ﺑﻪﻣﻨﻈﻮر ﺗﻮﻟﯿﺪ ﻣﻘﺎدﯾﺮ ﺑﺎﻻﯾﯽ از اﮐﺘﯿﻮﯾﻦ A اﺳﺘﻔﺎده ﺷﺪ. ﺑﻪاﯾﻦ ﻣﻨﻈﻮر اﺑﺘﺪاcDNA ﻧﺎﺣﯿﻪ ﺑﺎﻟﻎ ژن اﮐﺘﯿﻮﯾﻦ A ﺗﮑﺜﯿﺮ و در وﮐﺘﻮر )+(pET28a ﮐﻠﻮن ﺷﺪ. وﮐﺘﻮر ﺣﺎﺻﻞ ﺑﻪ ﺳﻮﯾﻪﻫﺎي )BL21(DE3) plysS ،BL21(DE3 وBL21(DE3) Rosetta gami اﻧﺘﻘﺎل داده ﺷﺪ. ﭘﺲ از اﻟﻘﺎي ﭘﺮوﻣﻮﺗﺮ ﺑﺎ اﺳﺘﻔﺎده از IPTG و ﺑﯿﺎن ﭘﺮوﺗﺌﯿﻦ، ﺗﻮﻟﯿﺪ اﮐﺘﯿﻮﯾﻦ A ﺑﻪ وﺳﯿﻠﻪ روشﻫﺎي SDS-PAGE و وﺳﺘﺮن ﺑﻼت ﺗﺄﯾﯿـﺪ ﺷـﺪ. ﻧﺘـﺎﯾﺞ ﺑﻪدﺳﺖ آﻣﺪه ﻧﺸﺎن داد ﮐﻪ ﺑﯿﺎن اﮐﺘﯿﻮﯾﻦ A در ﺳﯿﺘﻮﭘﻼﺳﻢ ﻫـﺮ ﺳـﻪ ﺳـﻮﯾﻪ روﯾﮑـﺮد ي ﻣـﺆﺛﺮ ﺑـﺮاي دﺳﺘﯿﺎﺑﯽ ﺑﻪ ﻣﯿﺰان ﺑﺎﻻﯾﯽ از ﭘﺮوﺗﺌﯿﻦ ﻧﻮﺗﺮﮐﯿﺐ اﺳﺖ اﻣـﺎ در اﯾـﻦ ﺑـﯿﻦ، ﺳـﻮﯾ ﻪ )BL21(DE3 ﻣﻘـﺪار ﺑﯿﺸﺘﺮي ﭘﺮوﺗﺌﯿﻦ ﺗﻮﻟﯿﺪ ﮐﺮده اﺳﺖ. در ﻣﺮﺣﻠﻪ ﺑﻌﺪ ﺑﻪﻣﻨﻈﻮر دﺳﺘﯿﺎﺑﯽ ﺑﻪ ﺷﮑﻞ ﻣﺤﻠﻮل اﮐﺘﯿـﻮﯾﻦ A از ﺑﯿﺎن ﻫﻤﺰﻣﺎن ﭼﭙﺮونﻫﺎي ﺳﯿﺘﻮﭘﻼﺳﻤﯽ GroEL/ES ،TF و DnaK/J ﺑﺎ وﮐﺘﻮر )+(pET28a ﮐﻪ ﺣﺎﻣﻞ اﮐﺘﯿﻮﯾﻦA ﺑﻮد، اﺳﺘﻔﺎده ﺷﺪ. ﻧﺘﺎﯾﺞ SDS-PAGE و وﺳﺘﺮن ﺑﻼت ﻧﺸﺎن داد ﮐﻪ ﺑﯿﺎن ﻫﻤﺰﻣﺎن اﮐﺘﯿﻮﯾﻦ A ﺑﺎ اﺳﺘﻔﺎده از ﭘﻼﺳﻤﯿﺪ ﭼﭙﺮوﻧﯽ pGro7 ﮐﻪ داراي ﭼﭙﺮونﻫـﺎي GroEL و GroES ﻣـﯽ ﺑﺎﺷـﺪ، درﺳﻮﯾﻪ )BL21(DE3 ﯾﮏ روﯾﮑﺮد ﻣﺆﺛﺮ ﺑﺮاي ﺗﻮﻟﯿﺪ ﭘﺮوﺗﺌﯿﻦ اﮐﺘﯿﻮﯾﻦ A ﻣﺤﻠﻮل اﺳﺖ.
چكيده لاتين :
Activin A, a member of the transforming growth factor-β (TGF-β)
superfamily, plays a central role in numerous physiological processes such
as cell differentiation, tissue repair, angiogenesis, differentiation of stem
cells, cell adhesion and apoptosis. Because of its various clinical usages,
recombinant production of it is beneficial. Since E. coli is one of the most
popular hosts for recombinant protein production, in this study, cytoplasmic
expression in this strain was used to produce high levels of Activin A. So,
the cDNA of the Activin A mature region was amplified and then cloned in
pET28a(+) vector. The resulting vector was transformed to BL21(DE3),
BL21(DE3)plysS, and BL21(DE3)Rosetta-gami strains. After induction the
promoter by using IPTG, Activin A production was confirmed by SDSPAGE
and Western blotting assays. The results showed that the expression
of Activin A in the cytoplasm of all three strains was an efficient approach to
obtain high levels of recombinant protein, but BL21(DE3) strain produced
more protein. At the next step in order to achieve soluble form of Activin A,
co-expression of cytoplasmic chaperones TF, GroEL/ES, and DnaK with
pET28a (+) vector was used. The SDS-PAGE and Western blotting results
showed that co-expression of Activin A with cytoplasmic plasmid pGro7
containing GroEL and GroES chaperones, in BL21(DE3) strain is an
efficient approach for producing of soluble Activin A.
