Mouse Peroxisomal Protein cDNA Cloning and Characterization of its Intraclleular Localization

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Mouse Peroxisomal Protein cDNA Cloning and

Characterization of its Intraclleular Localization

Somayeh Tanhaie, M.Sc.1, Kamran Ghaedi, Ph.D.1, 2*, Khadijeh Karbalaii, M.Sc.1,

Shanaz Razavi, Ph.D.3, Maryam Ostadsharif, M.Sc.1, 4, Maliheh Nazari-Jahantigh, B.Sc.1, 2,

Farzaneh Rabeei, B.Sc.1, Marziyeh Nematollahi, B.Sc.1, Hossein Baharvand, Ph.D.5,

Mohammad Hossein Nasr Esfahani, Ph.D.1*

1. Cell and Molecular Biology Department, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran 2. Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran

3. Anatomy Department, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 4. Basic Medical sciences Department, School of Midwifery and Nursery, Islamic Azad University

(Khorasgan Branch), Khorasgan, Iran

5. Stem Cells and Developmental Biology Department, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

* Corresponding Addresses: P.O.Box: 19395-4644, Cell and Molecular Biology Department, Royan Institute for Animal Bio-technology, ACECR, Isfahan, Iran

Email: kamranghaedi@royaninstitute.org Email: mh.nasr-esfahani@royaninstitute.org

Received: 3/Apr/2008, Accepted: 21/Sep/2008

Abstract

Objective: The aim of this study was to clone peroxisomal protein (PEP) cDNA in a mam-malian expression vector in a chimeric cDNA type, with enhanced green fluorescent protein (EGFP) cDNA. To investigate the intracellular localization of PEP protein linked to EGFP marker, the constructed plasmid was used for transfection into the chinese hamster ovary (CHO) cells.

Materials and Methods: Total RNA was extracted from the heart tissue of an adult mouse. PEP cDNA was constructed using reverse transcriptase and was amplified with specific prim-ers covering the entire length of ORF. RT-PCR products containing PEP cDNA were treated by enzymatic digestion and inserted into the pEGFP-C1 downstream of EGFP cDNA and were used for transformation into bacterial competent cells. The positive colonies which showed inserted PEP cDNA were selected for plasmid preparations and additional analysis was per-formed to ensure that PEP cDNA was inserted properly. Finally, to confirm the intracellular localization of EGFP-PEP, CHO cells were transfected with the constructed plasmid. Results: Our results confirmed amplification and cloning of the expected product. PEP cDNA encompasses 630 bp which encodes 209 amino acid residues. Bioinformatics analyses have shown the presence of a fibronectin type III domain (31-114 a.a.) and two hydrophobic do-mains (12-32 a.a. and 152-169 a.a., respectively). Because of the presence of serine, Lysine, leucine (SKI) in the C-terminal of the related protein, transfection data showed peroxisomal localization of PEP as was similar to the catalase.

Conclusion: Taken together these data showed that PEP is a peroxisomal protein. However the importance of its fibronectin type III and two hydrophobic domains should be assessed by further experiments.

Keywords: Fibronectin, Peroxisome, Peroxisome Targeting Signal 1

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ﻥﺁ

ﻰﻟﻮﻠﺳ

ﻥﻭﺭﺩ

ﻯﺮﻴﮔ

ﻯﺎﺟ

ﻰﺳﺭﺮﺑ

ﺵﻮﻣ

ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘ

ﻦﻴﺌﺗﻭﺮﭘ

cDNA

ﮓﻨﻴﻧﻮﻠﻛ

،4 ،1

،،M.Sc. ﻒﻳﺮﺷﺩﺎﺘﺳﺍﻢﻳﺮﻣ،

3

،،

Ph.D. ﻯﻮﺿﺭﺯﺎﻨﻬﺷ،1

،،M.Sc. ﻰﻳﻼﺑﺮﻛﻪﺠﻳﺪﺧ،

*2 ،1

،،

Ph.D. ﻯﺪﺋﺎﻗﻥﺍﺮﻣﺎﻛ،1

،،M.Sc. ﻰﻳﺎﻬﻨﺗﻪﻴﻤﺳ

،5

،،Ph.D. ﺪﻧﻭﺭﺎﻬﺑﻦﻴﺴﺣ،

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B.Sc.ﻰﻬﻠﻟﺍﺖﻤﻌﻧﻪﻴﺿﺮﻣ،1

،،

B.Sc.ﻰﻌﻴﺑﺭﻪﻧﺍﺯﺮﻓ،2 ،1

،،

B.Sc. ﻎﻴﺗﻥﺎﻬﺟﻯﺮﻈﻧﻪﺤﻴﻠﻣ

*1

،،Ph.D.ﻰﻧﺎﻬﻔﺻﺍﺮﺼﻧﻦﻴﺴﺣﺪﻤﺤﻣ

،ﻰﻟﻮﻜﻟﻮﻣﻭﻰﻟﻮﻠﺳﻰﺳﺎﻨﺷﺖﺴﻳﺯﻩﻭﺮﮔ،ﻰﻫﺎﮕﺸﻧﺍﺩﺩﺎﻬﺟﻰﻟﻮﻠﺳﻡﻮﻠﻋﺕﺎﻘﻴﻘﺤﺗﺰﻛﺮﻣ،ﻥﺎﻳﻭﺭﻩﺪﻜﺸﻫﻭﮋﭘ .1 ﻥﺍﺮﻳﺍ،ﻥﺎﻬﻔﺻﺍ

ﻥﺍﺮﻳﺍ،ﻥﺎﻬﻔﺻﺍ،ﻰﺳﺎﻨﺷﺖﺴﻳﺯﻩﻭﺮﮔ،ﻡﻮﻠﻋﻩﺪﻜﺸﻧﺍﺩ،ﻥﺎﻬﻔﺻﺍﻩﺎﮕﺸﻧﺍﺩ .2 ﻥﺍﺮﻳﺍ،ﻥﺎﻬﻔﺻﺍ،ﻰﻣﻮﺗﺎﻧﺁﻩﻭﺮﮔ،ﻰﻜﺷﺰﭘﻩﺪﻜﺸﻧﺍﺩ،ﻥﺎﻬﻔﺻﺍﻰﻜﺷﺰﭘﻡﻮﻠﻋﻩﺎﮕﺸﻧﺍﺩ .3

ﻥﺍﺮﻳﺍ،ﻥﺎﻬﻔﺻﺍ،ﻲﻜﺷﺰﭘﻪﻳﺎﭘﻡﻮﻠﻋﻩﻭﺮﮔ،ﻲﻳﺎﻣﺎﻣﻭﻱﺭﺎﺘﺳﺮﭘﻩﺪﻜﺸﻧﺍﺩ،ﻥﺎﮕﺳﺍﺭﻮﺧﺪﺣﺍﻭ،ﻲﻣﻼﺳﺍﺩﺍﺯﺁﻩﺎﮕﺸﻧﺍﺩ .4 ﻥﺍﺮﻳﺍ،ﻥﺍﺮﻬﺗ،ﻰﻨﻳﻮﻜﺗﻰﺳﺎﻨﺷﺖﺴﻳﺯﻭﻯﺩﺎﻴﻨﺑﻯﺎﻫﻝﻮﻠﺳﻩﻭﺮﮔ،ﻰﻫﺎﮕﺸﻧﺍﺩﺩﺎﻬﺟﻰﻟﻮﻠﺳﻡﻮﻠﻋﺕﺎﻘﻴﻘﺤﺗﺰﻛﺮﻣ،ﻥﺎﻳﻭﺭﻩﺪﻜﺸﻫﻭﮋﭘ .5

،ﻰﻫﺎﮕﺸﻧﺍﺩﺩﺎﻬﺟﻰﻟﻮﻠﺳﻡﻮﻠﻋﺕﺎﻘﻴﻘﺤﺗﺰﻛﺮﻣ،ﻥﺎﻳﻭﺭﻩﺪﻜﺸﻫﻭﮋﭘ،19395-4464 :ﻲﺘﺴﭘﻕﻭﺪﻨﺻ،ﻥﺍﺮﻳﺍ،ﻥﺎﻬﻔﺻﺍ :ﻝﻮﺌﺴﻣﻥﺎﮔﺪﻨﺴﻳﻮﻧﺱﺭﺩﺁ *

ﻰﻟﻮﻜﻟﻮﻣﻭﻰﻟﻮﻠﺳﻰﺳﺎﻨﺷﺖﺴﻳﺯﻩﻭﺮﮔ

Emails: kamranghaedi@royaninstitute.org :ﻚﻴﻧﻭﺮﺘﻜﻟﺍﺖﺴﭘ mh.nasr-esfahani@royaninstitute.org

87/6/31 :ﻪﻟﺎﻘﻣﺵﺮﻳﺬﭘ،87/1/15 :ﻪﻟﺎﻘﻣﺖﻓﺎﻳﺭﺩ

ﻩﺪﻴﻜﭼ

ﻥژﻥﺎﻴﺑﻱﻮﮕﻟﺍﻲﺳﺭﺮﺑﻭEGFP-C1ﻲﻧﺎﻴﺑﻲﺗﻮﻳﺭﺎﻛﻮﻳﺪﻴﻤﺳﻼﭘﺭﺩﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﻦﻴﺌﺗﻭﺮﭘcDNAﻥﺩﻮﻤﻧﻥﻮﻠﻛ :ﻑﺪﻫ * ﻱﺪﻨﻫﻪﭽﻛﻮﺧﻥﺍﺪﻤﺨﺗﻝﻮﻠﺳﺭﺩﺲﻧﺎﺳﺭﻮﻠﻓﺰﺒﺳﻦﻴﺌﺗﻭﺮﭘﺮﻛﺭﺎﻣﻪﺑﻞﺼﺘﻣ (Peroxisomal Protein) PEP ﻱﺎﻫﺮﻤﻳﺍﺮﭘﺯﺍﻩﺩﺎﻔﺘﺳﺍﺎﺑﻭﺪﺷﻪﻴﻬﺗﻪﻃﻮﺑﺮﻣcDNA،ﻎﻟﺎﺑﺵﻮﻣﺐﻠﻗﺯﺍﻝﻮﻠﺳﻞﻛRNAﺝﺍﺮﺨﺘﺳﺍﺯﺍﺲﭘ :ﺎﻫﺵﻭﺭﻭﺩﺍﻮﻣ *

ﺭﺍﺮﻗEGFP-C1ﺪﻴﻤﺳﻼﭘﺭﺩﻲﻤﻳﺰﻧﺁﻢﻀﻫﺯﺍﺲﭘﻩﺪﺷﺮﻴﺜﻜﺗPEP cDNA .ﺖﻓﺮﮔﺕﺭﻮﺻPEP cDNAﺮﻴﺜﻜﺗﻲﺻﺎﺼﺘﺧﺍ

ﺖﺸﻛﺯﺍﺲﭘﻭﺪﻳﺩﺮﮔﻡﺭﻮﻓﺲﻧﺍﺮﺗPEP cDNAﻭﺪﻴﻤﺳﻼﭘﻝﺎﺼﺗﺍﻝﻮﺼﺤﻣﺎﺑOne Shot TOP 10 ﻯﺎﻫﻱﺮﺘﻛﺎﺑ .ﺖﻓﺮﮔ ﺎﻬﻧﺁﺯﺍﻩﺪﺷﺺﻟﺎﺧﺪﻴﻤﺳﻼﭘﻱﻭﺭﺮﺑﻭﺪﻧﺪﻳﺩﺮﮔﺮﻴﺜﻜﺗﻭﺏﺎﺨﺘﻧﺍPCRﻥﻮﻣﺯﺁﺎﺑﺐﻴﻛﺮﺗﻮﻧﺪﻴﻤﺳﻼﭘﻱﺍﺭﺍﺩﺖﺒﺜﻣﻱﺎﻫﻲﻧﻮﻠﻛ 4 ﺎﺑﺎﻫﻝﻮﻠﺳﻦﻳﺍ،CHOﻯﺎﻫﻝﻮﻠﺳﻥﻭﺭﺩPEPﻥﺎﻴﺑﻱﻮﮕﻟﺍﻲﺳﺭﺮﺑﻱﺍﺮﺑ .ﺪﺷﻡﺎﺠﻧﺍﻲﻟﺍﻮﺗﻦﻴﻴﻌﺗﻭﻲﻤﻳﺰﻧﺁﻢﻀﻫﻱﺎﻫﺖﺴﺗ .ﺪﻧﺪﺷﺖﻜﻓﺲﻧﺍﺮﺗ 2000 ﻦﻴﻣﺎﺘﻜﻓﻮﭙﻴﻟﺮﺘﻴﻟﻭﺮﻜﻴﻣ 10 ﻭﺪﻴﻤﺳﻼﭘﻡﺮﮔﻭﺮﮕﻴﻣ

.ﺪﺷﺎﺑﻰﻣPEP cDNAﻥﺎﻤﻫﻩﺪﺷﻥﻮﻠﻛﻭﺮﻴﺜﻜﺗﻪﻌﻄﻗﻪﻛﺩﺮﻛﺖﺑﺎﺛﻲﻟﺍﻮﺗﻦﻴﻴﻌﺗﻭﻲﻤﻳﺰﻧﺁﻢﻀﻫﺞﻳﺎﺘﻧ :ﺎﻫﻪﺘﻓﺎﻳ *

ﻪﻨﻣﺍﺩﻚﻳﺩﻮﺟﻭﻩﺪﻨﻫﺩﻥﺎﺸﻧﻰﻜﻴﺗﺎﻣﺭﻮﻔﻧﺍﻮﻴﺑﻯﺎﻫﻰﺳﺭﺮﺑﻪﻛﺖﺳﺍﻪﻨﻴﻣﺁﺪﻴﺳﺍ 209 ﻩﺪﻨﻨﻛﺪﻛﻯﺯﺎﺑﺖﻔﺟ 630 cDNAﻦﻳﺍ 12 ﻪﻨﻴﻣﺁﺪﻴﺳﺍﺯﺍﺰﻳﺮﮔﺏﺁﻰﻳﺎﺸﻏﻥﻭﺭﺩﺲﻜﻴﻠﻫﺎﻔﻟﺁﻪﻨﻣﺍﺩﻭﺩﺩﺎﻳﺯﺭﺎﻴﺴﺑﻝﺎﻤﺘﺣﺍﺎﺑﻭ 114ﺎﺗ 31 ﻪﻨﻴﻣﺁﺪﻴﺳﺍﺯﺍ 3 ﻉﻮﻧﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓ ﻦﻳﺍﻪﻛﺪﻨﻛﻰﻣﺖﺑﺎﺛﺯﻻﺎﺗﺎﻛﻰﺻﺎﺼﺘﺧﺍﻯﺰﻴﻣﺁﮓﻧﺭﻩﺍﺮﻤﻫﻪﺑﻰﻟﻮﻠﺳﻦﺸﻜﻓﺲﻧﺍﺮﺗﺞﻳﺎﺘﻧ .ﺪﺷﺎﺑﻰﻣﻥﺁﺭﺩ 169 ﺎﺗ 152 ﻭ 32 ﺎﺗ .ﺩﻮﺷﻰﻣﻞﻘﺘﻨﻣﻡﻭﺰﻴﺴﻛﺍﺮﭘﻚﻣﺍﺪﻧﺍﻥﻭﺭﺩﻪﺑ،(Serine Lysine Lucine; SKL)،ﺩﻮﺧﻞﻴﺴﻛﻮﺑﺮﻛﻯﺎﻬﺘﻧﺍﻝﺎﻨﮕﻴﺳﺎﺑﻦﻴﺌﺗﻭﺮﭘ ﻪﻨﻣﺍﺩﻚﻳﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﺪﻨﭼﺮﻫ ؛ﺖﺳﺍﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘﻦﻴﺌﺗﻭﺮﭘﻚﻳPEPﻪﻛ ﺪﻨﻛﻰﻣ ﺕﺎﺒﺛﺍﺕﺎﻌﻟﺎﻄﻣ :ﻱﺮﻴﮔﻪﺠﻴﺘﻧ * .ﺖﺳﺍﺺﺨﺸﻣﺎﻧﺯﻮﻨﻫﺎﻬﻧﺁﺶﻘﻧﻪﻛﺩﺭﺍﺩﺰﻳﺮﮔﺏﺁﻪﻨﻣﺍﺩﻭﺩﻭ 3 ﻉﻮﻧﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓ

PTS1ﻝﺎﻨﮕﻴﺳ،ﻡﻭﺰﻴﺴﻛﺍﺮﭘ،ﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓ:ﻥﺎﮔژﺍﻭﺪﻴﻠﻛ *

196-203 :ﺕﺎﺤﻔﺻ،88 ﻥﺎﺘﺴﺑﺎﺗ،2 ﻩﺭﺎﻤﺷ،ﻢﻫﺩﺯﺎﻳﻝﺎﺳ،ﻪﺘﺧﺎﻳﻰﻜﺷﺰﭘﻪﻣﺎﻨﻠﺼﻓ

ﻪﻣﺪﻘﻣ

ﻲﻟﻮﻠﺳﻞﺧﺍﺩﻞﻜﺷﻱﻭﺮﻛﻰﻳﺎﺸﻏﻚﺗﻱﺎﻫﻚﻣﺍﺪﻧﺍﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻡﺎﻤﺗﺭﺩ ﻭ ﺪﻨﺷﺎﺑﻲﻣﺮﺘﻣﻭﺮﻜﻴﻣ ﻢﻫﺩ ﻚﻳﻰﻟﺍ ﻚﻳﺮﻄﻗ ﻪﺑ ﻪﻛﺪﻨﺘﺴﻫ ﻪﭼ ﺮﮔﺍ .(1) ﺪﻧﺭﺍﺩ ﺩﻮﺟﻭ ،ﻥﺎﺴﻧﺍﺎﺗ ﺮﻤﺨﻣﺯﺍ ،ﻲﺗﻮﻳﺭﺎﻛﻮﻳ ﻱﺎﻫﻝﻮﻠﺳ ﺯﺍ ﻮﻴﺗﺍﺪﻴﺴﻛﺍ ﻱﺎﻫﺱﺮﺘﺳﺍ ﻞﺑﺎﻘﻣ ﺭﺩ ﻉﺎﻓﺩ ﻭ ﻲﻳﺍﺩﺯﻢﺳ ﻱﺎﻫﺶﻨﻛﺍﻭ ﻞﻧﺎﮔﺭﺍﻦﻳﺍﺭﺩﺎﻣﺍﺪﺷﺎﺑﻲﻣﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘﻩﺪﺷﻪﺘﺧﺎﻨﺷﻝﺎﻤﻋﺍﻦﻳﺮﺗﻢﻬﻣ ﻪﻠﻤﺟﺯﺍﻲﻋﻮﻨﺘﻣﻚﻴﻟﻮﺑﺎﺘﻣﻱﺎﻫﺶﻨﻛﺍﻭ،ﻒﻠﺘﺨﻣﻱﺎﻫﻢﻳﺰﻧﺁﺩﻮﺟﻭﺎﺑ ﺰﺘﻨﺳﻮﻴﺑ ﻭ ﺮﻴﺠﻧﺯ ﺪﻨﻠﺑ ﺏﺮﭼ ﻱﺎﻫﺪﻴﺳﺍ ﺯﺍ ﻱﺭﺎﻴﺴﺑ ﻥﻮﻴﺳﺍﺪﻴﺴﻛﺍ ﺎﺘﺑ ﻱﺩﺮﻜﻠﻤﻋ ﺶﻘﻧ ﺖﻴﻤﻫﺍ .(2-5) ﺩﻮﺷﻲﻣ ﻡﺎﺠﻧﺍ ﺰﻴﻧ ﺎﻫﻥژﻮﻟﺎﻤﺳﻼﭘ ﺺﺨﺸﻣﺮﮔﻮﻟﺯﻡﻭﺭﺪﻨﺳﻞﻴﺒﻗﺯﺍﻲﻜﻴﺘﻧژﻱﺎﻫﻱﺭﺎﻤﻴﺑﺭﺩﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ .(3 ،2) ﺩﺩﺮﮔﻲﻣ ﻲﻜﻴﺘﻧژ ﻱﺎﻫﻱﺭﺎﻤﻴﺑ ﺯﺍﻰﻫﻭﺮﮔ ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﺰﻧژﻮﻴﺑ ﻱﺎﻫﻱﺭﺎﻤﻴﺑ ﺰﻧژﻮﻴﺑ ﻝﻮﺌﺴﻣ ﻱﺎﻫﻥژ ﺭﺩ ﻒﻠﺘﺨﻣ ﻯﺎﻫﺶﻬﺟ ﺎﻬﻧﺁ ﺭﺩ ﻪﻛ ﺪﻨﺷﺎﺑﻲﻣ

(3)

ﻡﻭﺩﻉﻮﻧﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﺏﺎﻳﻑﺪﻫﻝﺎﻨﮕﻴﺳﻡﺎﻧﻪﺑﻦﻴﺌﺗﻭﺮﭘﻲﻨﻴﻣﺁﻱﺎﻬﺘﻧﺍ .(15 ،12 ،11 ،6 ،3) ﺪﻨﺷﺎﺑﻰﻣ (PTS2) ﻰﻜﻳ (Peroxisomal Protein; PEP) ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﻦﻴﺌﺗﻭﺮﭘ ﻂﺳﻮﺗ 2002 ﻝﺎﺳﺭﺩﻪﻛﺖﺳﺍﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﺲﻜﻳﺮﺗﺎﻣﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘﺯﺍ ﺯﺍ .(16) ﺪﻳﺩﺮﮔﻥﻮﻠﻛﻭﻰﻳﺎﺳﺎﻨﺷﻥﺁﻲﻟﺍﻮﺗ ﺶﻧﺍﺭﺎﻜﻤﻫﻭﺰﻨﻴﺗﺭﺎﻣﺭﺮﻓ ﻰﻃﺭﺩﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻪﻛﺪﺳﺭﻰﻣﺮﻈﻧﻪﺑﻰﻠﺒﻗﺩﻭﺪﺤﻣﺕﻻﺎﻘﻣ ﻉﻮﻤﺠﻣ ﺕﺎﻌﻟﺎﻄﻣﺖﻬﺟﺍﺬﻟ .(16) ﺪﺷﺎﺑﻪﺘﺷﺍﺩﻰﻤﻬﻣﺶﻘﻧﻦﻴﻨﺟﻞﻣﺎﻜﺗﻭﺪﺷﺭ ﻦﻳﺍﻪﺑﻁﻮﺑﺮﻣcDNAﻪﻛﻢﻳﺪﺷﻥﺁﺮﺑ،ﻯﺩﺎﻴﻨﺑﻯﺎﻫﻝﻮﻠﺳﺭﺩﻯﺍﻪﻨﻴﻣﺯ EGFPﻥژﺖﺳﺩﻦﻴﻳﺎﭘpEGFP-C1ﻲﻧﺎﻴﺑﺭﻮﺘﻛﻭﺭﺩﺎﻤﻴﻘﺘﺴﻣﺍﺭﻥژ ﺭﺩﺲﭙﺳﻭﻥﻮﻠﻛ(Enhanced Green Flourescent Protein) .ﻢﻴﻳﺎﻤﻧﺖﻜﻓﺲﻧﺍﺮﺗ (Chinese Hamster Ovary)ﺍCHOﻝﻮﻠﺳ

ﺲﻧﺎﺳﺭﻮﻠﻓﺰﺒﺳ ﻦﻴﺌﺗﻭﺮﭘﻩﺪﻨﻨﻛﺪﻛﻥژ ﻯﺍﺭﺍﺩEGFP-C1ﺪﻴﻤﺳﻼﭘ ﻩﺪﺷﻥﻮﻠﻛﻥژ ﻝﻮﺼﺤﻣﻥﺁ ﻪﻄﺳﺍﻭ ﻪﺑﻥﺍﻮﺗﻰﻣﻰﺘﺣﺍﺭﻪﺑ ﻪﻛﺖﺳﺍ ﺭﺩﻥﺁ ﻯﺮﻴﮔﻯﺎﺟ ﻭ PEP ﻥژ ﻥﺎﻴﺑ ﺖﻳﺎﻬﻧ ﺭﺩ .ﺩﻮﻤﻧ ﻰﻳﺎﺳﺎﻨﺷ ﺍﺭ EGFP ﻯﻭﺎﺣ ﺐﻴﻛﺮﺗﻮﻧ ﻦﻴﺌﺗﻭﺮﭘ ﻥﺎﻴﺑ ﻚﻤﻛ ﻪﺑ ﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻢﻳﺰﻧﺁ ﻯﺍﺮﺑ ﻰﻤﻴﺷﻮﺘﻴﺳﻮﻧﻮﻤﻳﺍﻭ ﻥژ ﻦﻳﺍ ﺖﺳﺩ ﻻﺎﺑ ﺭﺩ ﻪﺘﻓﺮﮔ ﺭﺍﺮﻗ .ﺪﺷﻰﺳﺭﺮﺑﺯﻻﺎﺗﺎﻛﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘ

ﺎﻫ

ﺵﻭﺭ

ﺩﺍﻮﻣ

RNAﺝﺍﺮﺨﺘﺳﺍﻭﻲﻧﺍﻮﻴﺣﻝﺪﻣ ﺖﻓﺎﺑ،ﻯﺭﻮﺳﺩﺍﮋﻧﺵﻮﻣﻚﻳﺯﺍ ﺐﻠﻗﺖﻓﺎﺑﻯﺯﺎﺳﺍﺪﺟﺯﺍ ﺲﭘ .ﺪﺷﻩﺩﺍﺩ ﻮﺷﻭﺖﺴﺷﺩﺮﺳﻱژﻮﻟﻮﻳﺰﻴﻓ ﻡﺮﺳﺎﺑ ﻭ ﺪﻳﺩﺮﮔﻪﻌﻄﻗﻪﻌﻄﻗ

،ﻥژﺎﻨﻴﺳ) RNX-PLUS ﻝﻮﻠﺤﻣ ﺯﺍ ﺮﺘﻴﻟﻲﻠﻴﻣ ﻚﻳﻰﺘﻓﺎﺑ ﺕﺎﻌﻄﻗﻪﺑ ﺝﺍﺮﺨﺘﺳﺍﺭﻮﻛﺬﻣﺖﻴﻛﻪﻠﻴﺳﻭﻪﺑ ﻲﻟﻮﻠﺳRNA ﻞﻛﻭﻪﻓﺎﺿﺍ (ﻥﺍﺮﻳﺍ ﻱﺮﺘﻣﻮﺘﻓﻭﺮﺘﻜﭙﺳﺍ ﻩﺎﮕﺘﺳﺩ ﺎﺑ ﻩﺪﺷ ﺺﻴﻠﺨﺗ RNA ﺖﻈﻠﻏ .ﺪﺷ ﺩﺭﻮﻣ ﺰﻴﻧ ﻲﻘﻓﺍ ﺯﺭﻮﻓﻭﺮﺘﻜﻟﺍ ﺎﺑ ﻩﺪﺷ ﺺﻴﻠﺨﺗ RNA ﻭ ﻰﺑﺎﻳﺯﺭﺍ .ﺖﻓﺮﮔﺭﺍﺮﻗ ﻰﺳﺭﺮﺑ

RT-PCRﻭcDNAﺰﺘﻨﺳ ﻪﺑ ﻰﮔﺩﻮﻟﺁ ﻑﺬﺣ ﺖﻬﺟ ﻩﺪﺷ ﺝﺍﺮﺨﺘﺳﺍ RNA ﻡﺮﮔﻭﺮﻜﻴﻣ 2 ﺭﺩﻪﻘﻴﻗﺩ30 ﺕﺪﻣﻪﺑDNase I (Fermentas)ﻢﻳﺰﻧﺁﺎﺑﻰﻣﻮﻧژDNA ﺖﻴﻛﺯﺍﻩﺩﺎﻔﺘﺳﺍﺎﺑ cDNAﺰﺘﻨﺳﻱﺍﺮﺑﻥﺁﺯﺍﻭﺭﺎﻤﻴﺗﺩﺍﺮﮔﻲﺘﻧﺎﺳﻪﺟﺭﺩ37 ﻪﺑ dTﻭ (Fermentas) ﻱﺎﻫﺮﻣﻮﮕﻴﻟﺍﻭ cDNAﻭ (Fermentas) ﺰﺘﻨﺳ

ﺯﺍﺮﺘﻴﻟﻭﺮﻜﻴﻣﻭﺩﺎﺑPCRﺶﻨﻛﺍﻭﻥﺁﺯﺍﺲﭘ .ﺪﺷﻩﺩﺎﻔﺘﺳﺍﺮﻤﻳﺍﺮﭘﻥﺍﻮﻨﻋ pep-Rﻭpep-F Bgl IIﻱﺎﻫﺮﻤﻳﺍﺮﭘﺭﻻﻮﻣﻮﻜﻴﭘ 5 ،ﻩﺪﺷﺰﺘﻨﺳ cDNA

Pfu (Fermentas)ﻢﻳﺰﻧﺁﻲﻤﻳﺰﻧﺁﺪﺣﺍﻭ 1/25 ﻭ (1 ﻝﻭﺪﺟ) Sal I 35 ﺲﭙﺳﻭﺩﺍﺮﮔﻲﺘﻧﺎﺳﻪﺟﺭﺩ 94 ﻪﻘﻴﻗﺩ 4 ﻪﻴﻟﻭﺍﻥﻮﻴﺳﺍﺭﻮﺗﺎﻧﺩﻪﻣﺎﻧﺮﺑﺎﺑ

ﻪﺟﺭﺩ 60 ،ﻪﻘﻴﻗﺩ 1ﺕﺪﻣ ﻪﺑ ﺩﺍﺮﮔﻲﺘﻧﺎﺳ ﻪﺟﺭﺩ 94 ﺐﻴﺗﺮﺗ ﻪﺑ ﻞﻜﻴﺳ ﻭﻪﻘﻴﻗﺩ 1ﺕﺪﻣﻪﺑﺩﺍﺮﮔﻲﺘﻧﺎﺳﻪﺟﺭﺩ 72 ،ﻪﻘﻴﻗﺩ 1ﺕﺪﻣﻪﺑﺩﺍﺮﮔﻲﺘﻧﺎﺳ ﺪﺷﻡﺎﺠﻧﺍﻪﻘﻴﻗﺩ 10ﺕﺪﻣﻪﺑﺩﺍﺮﮔﻲﺘﻧﺎﺳﻪﺟﺭﺩ 72ﻰﻧﺎﻳﺎﭘﻪﻠﺣﺮﻣﻡﺎﺠﻧﺍﺮﺳ PCRﺶﻨﻛﺍﻭ ﻥﺎﻣﺯﻢﻫ .(Thermal cycler Eppendorf ﻩﺎﮕﺘﺳﺩ) ﻭβ Tubulin Fﻰﺻﺎﺼﺘﺧﺍﻯﺎﻫﺮﻤﻳﺍﺮﭘﺎﺑ beta -Tubulin 5 ﻥژﻯﺍﺮﺑ ﻡﺎﺠﻧﺍcDNAﺰﺘﻨﺳﺖﺤﺻﺪﻴﻳﺎﺗﺭﻮﻈﻨﻣﻪﺑ (1 ﻝﻭﺪﺟ) β Tubulin R ﺯﺭﻮﻓﻭﺮﺘﻜﻟﺍ ﺪﺻﺭﺩ1 ﺯﻭﺭﺎﮔﺁ ﻝژ ﻱﻭﺭ ﺮﺑ PCR ﺕﻻﻮﺼﺤﻣ .ﺪﺷ .ﺪﻧﺪﺷ

ﻲﻤﻳﺰﻧﺁﻢﻀﻫ ﻝﻮﺼﺤﻣ ﻥﺍﻮﻨﻋ ﻪﺑ ﻪﺘﻓﺎﻳ ﺮﻴﺜﻜﺗPEP cDNA ﻪﻌﻄﻗ ﺪﻴﻳﺎﺗ ﻱﺍﺮﺑ ﻩﺩﺎﻔﺘﺳﺍPvu IIﻭ (Fermentas)ﻢﻳﺰﻧﺁﺯﺍ،RT-PCRﺶﻨﻛﺍﻭﻲﻳﺎﻬﻧ

.ﺪﺷ

EGFP-PEPﻞﻣﺎﺣﻲﻧﺎﻴﺑﺪﻴﻤﺳﻼﭘﺖﺧﺎﺳ PCR ﺶﻨﻛﺍﻭﺭﺩ ﻪﻛPEP cDNAﻪﺑ ﻁﻮﺑﺮﻣPCRﻝﻮﺼﺤﻣ ﻭ ﺍﺪﺘﺑﺍﺭﺩ pep-R Sal I ﻭ pep-F Bgl II ﻱﺎﻫﺮﻤﻳﺍﺮﭘ ﻪﻄﺳﺍﻭ ﻪﺑ ﻭ Bgl II ﻢﻳﺰﻧﺁ ﻭﺩ ﺵﺮﺑ ﻱﺎﻫﻩﺎﮕﻳﺎﺟﻞﻣﺎﺣ ﺐﻴﺗﺮﺗ ﻪﺑ ﺩﻮﺧ ﻱﺎﻬﺘﻧﺍ ﻭ Bgl II (Fermentas) ﻱﺎﻫﻢﻳﺰﻧﺁ ﻂﺳﻮﺗ ،ﺩﻮﺑ ﻩﺪﻳﺩﺮﮔ Sal I ﻲﻧﺎﻴﺑ ﺪﻴﻤﺳﻼﭘ ﺭﺩ ﺺﻴﻠﺨﺗ ﺯﺍﺲﭘﻭ ﻪﺘﻓﺎﻳ ﺵﺮﺑSal I (TaKaRa) ﺭﺍﺮﻗ ﺭﻮﻛﺬﻣ ﻱﺎﻫﻢﻳﺰﻧﺁ ﻩﺎﮕﻳﺎﺟ ﺭﺩ pEGFP-C1ﻭ (Clontech)

-Ligaﺖﻴﻛﺎﺑﻰﻧﺎﻴﺑﺪﻴﻤﺳﻼﭘﻪﺑﻪﺘﻓﺎﻳﺮﻴﺜﻜﺗﻪﻌﻄﻗﻝﺎﺼﺗﺍﻞﻤﻋ .ﺖﻓﺮﮔ ﻱﺎﻫﻱﺮﺘﻛﺎﺑ .ﺪﺷﻡﺎﺠﻧﺍﺖﻴﻛ ﻞﻜﺗﻭﺮﭘ ﻖﺒﻃTaKaRa ﺖﻛﺮﺷ tion One Shot TOP 10 Chemically competent ﻭ(Invitrogen)

ﻡﺭﻮﻔﺴﻧﺍﺮﺗ ﻪﻠﺻﺎﺣ ﺪﻴﻤﺳﻼﭘ ﺎﺑ ﻲﺗﺭﺍﺮﺣ ﻙﻮﺷ ﺵﻭﺭ ﻪﺑ E.Coli

.ﺪﻧﺪﻳﺩﺮﮔ ﻦﻴﺴﻳﺎﻣﺎﻧﺎﻛﻯﻭﺎﺣ2YTﺖﺸﻛﻂﻴﺤﻣﻱﻭﺭﺮﺑﻩﺩﺮﻛﺪﺷﺭﻱﺎﻫﻲﻧﻮﻠﻛﻦﻴﺑﺯﺍ ﺎﺑﺪﻴﻤﺳﻼﭘﻯﻭﺎﺣﻯﺎﻫﻰﻧﻮﻠﻛ،(GIBCO)ﺮﺘﻴﻟﻲﻠﻴﻣﺮﻫﺭﺩﻡﺮﮔﻭﺮﻜﻴﻣ 30

pep-R Sal Iﻭpep-F Bgl II ﻱﺎﻫﺮﻤﻳﺍﺮﭘﺎﺑ PCR ﺶﻨﻛﺍﻭ ﺯﺍ ﻩﺩﺎﻔﺘﺳﺍ .ﺪﻧﺪﺷﺏﺎﺨﺘﻧﺍ

DNAﻲﻟﺍﻮﺗﻦﻴﻴﻌﺗ 4 ﺯﺍﻩﺩﺎﻔﺘﺳﺍﺎﺑﻲﻟﺍﻮﺗﻦﻴﻴﻌﺗﻯﺍﺮﺑcDNA PEPﻱﻭﺎﺣﻰﻧﻮﻠﻛﻭﺩ

،pep-F Bgl II ،pep-R Sal I ،EGFP-C1 F ،EGFP-C1 Rﺮﻤﻳﺍﺮﭘ

.ﺪﻳﺩﺮﮔﻝﺎﺳﺭﺍﺖﺴﻳﺯﻮﭘﺎﻜﺗﺖﻛﺮﺷﻪﺑ

.ﺪﺷﺎﺑﻲﻣPEP cDNAﻲﻟﺍﻮﺗﺎﺑﻖﺒﻄﻨﻣﺖﺳﺍﻩﺪﺷﺺﺨﺸﻣﻂﺧﺮﻳﺯﺎﺑﻪﻛﻲﺘﻤﺴﻗ :1 ﻝﻭﺪﺟ

ﺮﻤﻳﺍﺮﭘﻡﺎﻧ ﺮﻤﻳﺍﺮﭘﻲﻟﺍﻮﺗ

ﻢﻴﺨﺿﻑﻭﺮﺣ

pep-F Bgl II

5'-ATT AGA TCT CCC CCA GGG CCG TGC GCC-3'

Bgl IIﻢﻳﺰﻧﺁﺵﺮﺑﻩﺎﮕﻳﺎﺟ

pep-R Sal I

5'-AAA AGT CGA CTC ATA TCT TGC TGC GGA GGA GA-3'

Sal I ﻢﻳﺰﻧﺁﺵﺮﺑﻩﺎﮕﻳﺎﺟ

EGFP-C1 F 5'-AAC GAG AAG CGC GAT CAC ATG G-3'

EGFP-C1 R 5'-GCA ATA AAC AAG TTA ACA ACA AC-3'

β tubulin F 5'-TCA CTG TGC CTG AAC TTA CC-3'

β tubulin R 5'-GGA ACA TAG CCG TAA ACT G C-3'

(4)

ﺮﻤﻳﺍﺮﭘﻲﺣﺍﺮﻃ

cDNA PEP) ﻱﺎﻫﻲﻟﺍﻮﺗ ﻪﺑ ﻪﺟﻮﺗ ﺎﺑ ﺮﻤﻳﺍﺮﭘ ﻲﺣﺍﺮﻃ Clontech, catalog)ﻭ EGFP-C1 ﺪﻴﻤﺳﻼﭘ ،(NM_027402

β-tubulin5) ﻭ(GenBank Accession #: U55763) ﻭ(#6084-1

Oligo 6.2 ﺭﺍﺰﻓﺍﻡﺮﻧ ﺯﺍ ﻩﺩﺎﻔﺘﺳﺍ ﺎﺑ 5 cDNA (NM_011655O (http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm) ﻪﻄﺳﺍﻭ ﺎﺑ Bioneer ﺖﻛﺮﺷ ﺯﺍ ﻩﺪﺷ ﺰﺘﻨﺳ ﻯﺎﻫﺮﻤﻳﺍﺮﭘ .ﺪﺷ ﻡﺎﺠﻧﺍ ﻱﺎﻫﺪﻴﺳﺍ ﻲﻟﺍﻮﺗ ﺰﻴﻟﺎﻧﺁ ﻱﺍﺮﺑ .ﺪﺷ ﻱﺭﺍﺪﻳﺮﺧ ﺖﺴﻳﺯﻮﭘﺎﻜﺗ ﺖﻛﺮﺷ Bio Edit Version 7.0.4 ﺭﺍﺰﻓﺍﻡﺮﻧ ﺯﺍ PEP ﻦﻴﺌﺗﻭﺮﭘ ﻪﻨﻴﻣﺁ (http://www.mbio.ncsu.edu/BioEdit/bioedit.htm ) ﺯﺍﻥﺁﻲﻜﻴﺗﺎﻣﺭﻮﻔﻧﺍﻮﻴﺑﺰﻴﻟﺎﻧﺁﻭﻲﻨﻴﺌﺗﻭﺮﭘﺭﺎﺘﺧﺎﺳﺮﺘﺸﻴﺑﻲﺳﺭﺮﺑﻱﺍﺮﺑﻭ http://www.ch.embnet.org/software/)ﺍ TMpred ﻱﺎﻫﺭﺍﺰﻓﺍﻡﺮﻧ

http://smart.embl-heidel-)ﺍ SMART ﻭ (TMPRED_from.html

.ﺪﺷﻩﺩﺎﻔﺘﺳﺍ(berg.de/e

ﺎﻫﻝﻮﻠﺳ ﻥﺪﺷﺖﻜﻓﺲﻧﺍﺮﺗﻭﻭﻲﻟﻮﻠﺳﺖﺸﻛ

.ﺪﻳﺩﺮﮔ ﻪﻴﻬﺗ ﻥﺍﺮﻬﺗ ﻥﺎﻳﻭﺭ ﻩﺪﻜﺸﻫﻭﮋﭘ ﺯﺍ CHO ﻲﻟﻮﻠﺳ ﻩﺩﺭ ﻱﻭﺎﺣHam'sF12 (Sigma) ﺖﺸﻛﻂﻴﺤﻣﺭﺩCHO ﻱﺎﻫﻝﻮﻠﺳ ﻱﺎﻣﺩ ﻭ CO2 ﺪﺻﺭﺩ5 ﺎﺑﺭﻮﺗﺎﺑﻮﻜﻧﺍ ﺭﺩ (Gibco) ﺪﺻﺭﺩ10 FCS

ﺯﺍ ﺲﭘ .ﺪﺷﻩﺩﺍﺩﺖﺸﻛﺪﺻﺭﺩ95 ﺖﺑﻮﻃﺭﻭﺩﺍﺮﮔﻲﺘﻧﺎﺳﻪﺟﺭﺩ 37 -Try/EDTA (Gib)ﻚﻤﻛﻪﺑﺎﻫﻝﻮﻠﺳ،ﻚﺳﻼﻓﻒﻛﻥﺪﺷﺵﻭﺮﻔﻣ ﻪﻧﺎﺧﺮﻫﺭﺩﻝﻮﻠﺳ 9×104ﺩﺍﺪﻌﺗ،ﻰﻟﻮﻠﺳﺵﺭﺎﻤﺷﺯﺍﺲﭘﻭﻪﻨﻴﺴﭙﻳﺮﺗ

co (ﺮﺘﻣﻲﻠﻴﻣ 24×24 ﻞﻣﻻ) ﻯﺭﺍﺬﮔﻞﻣﻻ ﻞﺒﻗﺯﺍﻪﻛﻪﻧﺎﺧ 6 ﻯﺎﻫﺶﻳﺩ ﺪﺻﺭﺩ 50-60 ﺎﻫﻝﻮﻠﺳﻪﻜﻨﻳﺍﺯﺍ ﺲﭘ .ﺪﺷﻩﺩﺍﺩﺖﺸﻛ،ﺪﻧﺩﻮﺑﻩﺪﺷ Opti-MEMﻝﻮﻠﺤﻣﺮﺘﻴﻟﻭﺮﻜﻴﻣ 250 ﺍﺪﺘﺑﺍ ،ﺪﻧﺩﺮﻛﺮﭘﺍﺭﺶﻳﺩﻒﻛ (ﺪﻴﻤﺳﻼﭘﻝﻮﻠﺤﻣ) EGFP-PEP ﺪﻴﻤﺳﻼﭘ ﻡﺮﮔﻭﺮﻜﻴﻣ 4 ﻯﻭﺎﺣ I ﺮﺘﻴﻟﻭﺮﻜﻴﻣ 10 ﻯﻭﺎﺣ Opti-MEM I ﻝﻮﻠﺤﻣ ﺮﺘﻴﻟﻭﺮﻜﻴﻣ 250 ﻭ ﻪﻴﻬﺗ (ﻦﻴﻣﺎﺘﻜﻓﻮﭙﻴﻟﻝﻮﻠﺤﻣ) Lipofectamine 2000 (Invitrogen) ،ﻪﻘﻴﻗﺩ 5 ﺕﺪﻣﻪﺑﻕﺎﺗﺍﻯﺎﻣﺩﺭﺩﻝﻮﻠﺤﻣﻭﺩﻥﻮﻴﺳﺎﺑﻮﻜﻧﺍﺯﺍﺲﭘ .ﺪﺷ ﻝﻮﻠﺤﻣ ﻭ (ﺖﻜﻓﺲﻧﺍﺮﺗﻝﻮﻠﺤﻣ) ﻁﻮﻠﺨﻣﺮﮕﻳﺪﻜﻳ ﺎﺑﻝﻮﻠﺤﻣ ﻭﺩﻦﻳﺍ ﻂﻴﺤﻣﺲﭙﺳ .ﺪﺷﻪﺑﻮﻜﻧﺍﻕﺎﺗﺍﻯﺎﻣﺩﺭﺩﺮﮕﻳﺩﻪﻘﻴﻗﺩ 20 ﺕﺪﻣﻪﺑﻪﻠﺻﺎﺣ ﺖﺸﻛ ﻂﻴﺤﻣﺎﺑﻰﻣﺍﺭﺁ ﻪﺑ ﺎﻫﻝﻮﻠﺳﻭ ﺪﻳﺩﺮﮔﻑﺬﺣﺎﻫﻝﻮﻠﺳﺖﺸﻛ ﻩﺩﺍﺩﻮﺷﻭﺖﺴﺷﻚﻴﺗﻮﻴﺑﻰﺘﻧﺁﻭﻡﺮﺳﺪﻗﺎﻓOpti-MEM I (Gibco) ﻂﻴﺤﻣ ﺮﺘﻴﻟﻲﻠﻴﻣ 2 ﺎﺑ ﻩﺍﺮﻤﻫ ﺖﻜﻓﺲﻧﺍﺮﺗ ﻝﻮﻠﺤﻣ ﺖﻳﺎﻬﻧ ﺭﺩ .ﺪﺷ ﻪﺑ ﻂﻴﺤﻣ ﻦﻳﺍ ﺎﺑ ﺎﻫﻝﻮﻠﺳ .ﺪﺷ ﻪﻓﺎﺿﺍ ﻪﻧﺎﺧ ﺮﻫ ﻪﺑ Opti-MEM I ﺪﺻﺭﺩ95 ،ﺩﺍﺮﮔﻰﺘﻧﺎﺳ ﻪﺟﺭﺩ 37 ﺭﻮﺗﺎﺑﻮﻜﻧﺍ ﺭﺩ ﺖﻋﺎﺳ 3 ﺕﺪﻣ

ﻂﻴﺤﻣ ﻥﺁ ﺯﺍ ﺲﭘ .ﺪﺷ ﻩﺩﺍﺩ ﺖﺸﻛ CO2 ﺪﺻﺭﺩ 5 ﻭ ﺖﺑﻮﻃﺭ

ﻪﻧﺎﺧﺮﻫ ﻪﺑ CHOﻝﻮﻠﺳ ﻝﻮﻤﻌﻣ ﺖﺸﻛ ﻂﻴﺤﻣﻭ ﻑﺬﺣﺖﻜﻓﺲﻧﺍﺮﺗ 48 .ﺪﻳﺩﺮﮔ ﻞﻘﺘﻨﻣﺭﻮﺗﺎﺑﻮﻜﻧﺍ ﻪﺑ ﺖﺸﻛﻪﻣﺍﺩﺍﻯﺍﺮﺑ ﺎﻫﻝﻮﻠﺳﻭﻪﻓﺎﺿﺍ ﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﻥژﻥﺎﻴﺑ ﻰﺳﺭﺮﺑﺭﻮﻈﻨﻣﻪﺑ ،ﺖﻜﻓﺲﻧﺍﺮﺗﺯﺍ ﺲﭘﺖﻋﺎﺳ ﺎﺑ ﺎﻫﻝﻮﻠﺳ ﻦﻳﺍ ،ﻩﺪﺷ ﺖﻜﻓﺲﻧﺍﺮﺗ ﻯﺎﻫﻝﻮﻠﺳ ﺭﺩ EGFP-PEP ﻰﺑﺎﻳﺩﺭ ﺖﻬﺟ ﻭ ﺲﻜﻴﻓ (Sigma) ﺪﺻﺭﺩ 4 ﺪﻴﺋﺪﻟﺎﻣﺮﻓﺍﺭﺎﭘ ﻝﻮﻠﺤﻣ ﻰﻤﻴﺷﻮﺘﻴﺳﻮﻨﻤﻳﺍ ،ﺭﻮﻛﺬﻣ ﻯﺎﻫﻝﻮﻠﺳﺭﺩ ﺯﻻﺎﺗﺎﻛﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﺮﻛﺭﺎﻣ -Rabbit Anti Cata ﻭ(abcam 1:300)ﻰﺻﺎﺼﺘﺧﺍ ﻯﺩﺎﺑﻰﺘﻧﺁ ﺎﺑ

Donkey Anti ﻭ(Amersham 1:400)ﻪﻳﻮﻧﺎﺛﻯﺩﺎﺑﻰﺘﻧﺁﻭ lase پﻮﻜﺳﻭﺮﻜﻴﻣﺯﺍﻩﺩﺎﻔﺘﺳﺍ ﺎﺑﺞﻳﺎﺘﻧ .ﺪﺷﻡﺎﺠﻧﺍ Rabbit Texas Red .ﺪﺷﻩﺪﻫﺎﺸﻣOlympus BX5ﺖﻨﺳﺭﻮﻠﻓ .ﺖﺳﺍﻥﺎﻳﻭﺭﻩﺪﻜﺸﻫﻭﮋﭘﻕﻼﺧﺍﻪﺘﻴﻤﻛﻪﺑﻮﺼﻣﺡﺮﻃﻦﻳﺍ

ﺎﻫ

ﻪﺘﻓﺎﻳ

RT-PCRﻭﻝﻮﻠﺳﻲﻠﻛRNAﻥﺩﻮﻤﻧﺍﺪﺟ ﺭﺩ .ﺪﻫﺩﻰﻣﻥﺎﺸﻧﺍﺭﺐﻠﻗﺖﻓﺎﺑﺯﺍﻩﺪﺷﺝﺍﺮﺨﺘﺳﺍRNA،1Aﻞﻜﺷ 28S,18S and 5S ribosomalﻯﺎﻫﺪﻧﺎﺑﻩﺪﺷﺝﺍﺮﺨﺘﺳﺍﻪﻧﻮﻤﻧﻭﺩﺮﻫ

ﻩﺪﻨﻫﺩﻥﺎﺸﻧﺮﮕﻳﺩﻯﺎﻫﺪﻧﺎﺑﺩﻮﺟﻭﻡﺪﻋﻭﺩﻮﺷﻰﻣﻩﺪﻳﺩﺡﻮﺿﻭﻪﺑRNA DNAﺮﮕﻧﺎﺸﻧﺩﻮﺟﻭﺎﻨﻤﺿ .ﺪﺷﺎﺑﻰﻣﻩﺪﺷﺺﻴﻠﺨﺗRNAﻯﻻﺎﺑﺖﻴﻔﻴﻛ ﺩﺭﻮﻣﻝژﻱﻻﺎﺑﺖﻴﻔﻴﻛﻥﺩﺍﺩﻥﺎﺸﻧﺭﻮﻈﻨﻣﻪﺑﺎﻬﻨﺗ RNAﺭﺎﻨﻛﺭﺩﻝژﺭﺩ .ﺩﺭﺍﺪﻧﻱﺍﻪﺴﻳﺎﻘﻣﻪﺒﻨﺟﻭﺖﺳﺍﻩﺪﺷﻪﺘﻓﺮﮔﺭﺍﺮﻗﻩﺩﺎﻔﺘﺳﺍ ﻥﺎﻴﺑ ﻻﺎﻤﺘﺣﺍ 1 ﻥﻮﺘﺳﺭﺩ ﻱﺯﺎﺑ ﺖﻔﺟ 630 ﺪﻧﺎﺑ ،1B ﻞﻜﺷ ﺭﺩ RNA ﺯﺍ ﻩﺪﺷﺰﺘﻨﺳ cDNA ﻯﻮﮕﻟﺍ ﺎﺑ PEPcDNA ﺮﻴﺜﻜﺗ ﻩﺪﻨﻨﻛ

ﻝﺮﺘﻨﻛﻪﻧﻮﻤﻧﻥﺍﻮﻨﻋﻪﺑ ﺮﻄﻘﻣﺏﺁ ﺯﺍﺶﻨﻛﺍﻭﻦﻳﺍﺭﺩ .ﺪﺷﺎﺑﻰﻣﻰﺘﻓﺎﺑ ﻢﻳﺰﻧﺁﻥﺁﻪﺑcDNAﺰﺘﻨﺳﻪﻠﺣﺮﻣﺭﺩﻪﻛﻯﺍﻪﻧﻮﻤﻧﺯﺍﻭ (2 ﻥﻮﺘﺳ) ﻰﻔﻨﻣ ﻲﻔﻨﻣ RT ﻥﺍﻮﻨﻋ ﻪﺑ ﺩﻮﺑ ﻩﺪﺸﻧ ﻪﻓﺎﺿﺍReverse Transcriptase ﻭﺩ ﻦﻳﺍ ﺭﺩPCR ﺯﺍ ﺲﭘﺪﻧﺎﺑ ﺩﻮﺟﻭ ﻡﺪﻋ .(3 ﻥﻮﺘﺳ) ﺪﺷ ﻩﺩﺎﻔﺘﺳﺍ ﺯﺍPEPﻰﻟﺎﻤﺘﺣﺍﺮﻴﺜﻜﺗﻡﺪﻋﻭﻰﮔﺩﻮﻟﺁﻡﺪﻋﺮﮕﻧﺎﻴﺑﺐﻴﺗﺮﺗﻪﺑﻪﻧﻮﻤﻧ ﻞﻜﺷ 4 ﻥﻮﺘﺳﺭﺩﺯﺎﺑﺖﻔﺟ 318 ﺪﻧﺎﺑ .ﺪﺷﺎﺑﻰﻣﻰﻣﻮﻧژDNAﻯﻭﺭ ﻩﺩﺎﻔﺘﺳﺍ ﺎﺑcDNA β-tubulin 5 ﺯﺍ ﻯﺍﻪﻌﻄﻗPCR ﻪﺑﻁﻮﺑﺮﻣ ،1B ﺖﺤﺻ ﻩﺪﻨﻫﺩ ﻥﺎﺸﻧ ﻪﻛ ﺖﺳﺍ ﻦﻴﻟﻮﺑﻮﺗﺎﺘﺑ ﻲﺻﺎﺼﺘﺧﺍ ﻱﺎﻫﺮﻤﻳﺍﺮﭘ ﺯﺍ .ﺪﺷﺎﺑﻰﻣ ﺭﺎﻛ ﻞﺣﺍﺮﻣ ﺖﻴﻔﻴﻛﻭ ﺎﺑ PEP cDNAﻪﺑﻁﻮﺑﺮﻣPCRﻝﻮﺼﺤﻣﺵﺮﺑﻪﺠﻴﺘﻧ،1C ﻞﻜﺷ ﺩﺎﺠﻳﺍﺯﺎﺑﺖﻔﺟ150ﻭ 286 ﻭ 191 ﺕﺎﻌﻄﻗ .ﺪﻫﺩﻰﻣﻥﺎﺸﻧﺍﺭPvu IIﻢﻳﺰﻧﺁ ﺵﺮﺑﻩﺎﮕﻳﺎﺟﻭﺩﺖﺴﻜﺷﺯﺍﻞﺻﺎﺣﺭﺎﻈﺘﻧﺍﺩﺭﻮﻣﺕﺎﻌﻄﻗﻖﺑﺎﻄﻣﺎﻘﻴﻗﺩﻩﺪﺷ .ﺪﺷﺎﺑﻲﻣ PEPﻡ cDNAﺭﺩPvu IIﻢﻳﺰﻧﺁ

Ladder 1 2

28S

18S

5S

A

Ladder 1 2 3 4

1000 646 (bp)

318 (bp)

B

Ladder Digested

pep

286 bp300 bp

191 bp 200 bp 150 bp

100 bp

C

ﻪﻧﻮﻤﻧ .2 ﻭ 1 ﻩﺭﺎﻤﺷﻪﻧﻮﻤﻧ .1 ،ﺯﺎﺘﻨﻣﺮﻓﺯﺎﺑﺖﻔﺟ 100 ﺮﻛﺭﺎﻣ .ﺵﻮﻣﺐﻠﻗﺖﻓﺎﺑﺯﺍRNAﺝﺍﺮﺨﺘﺳﺍﺞﻳﺎﺘﻧ،ﺐﻠﻗﺖﻓﺎﺑﺯﺍRNAﺝﺍﺮﺨﺘﺳﺍ .A :1 ﻞﻜﺷ

ﻝﺮﺘﻨﻛﻪﻧﻮﻤﻧ .3،RTﻪﻧﻮﻤﻧ .2،PEPﻲﺻﺎﺼﺘﺧﺍﻱﺎﻫﺮﻤﻳﺍﺮﭘﺎﺑcDNAﺮﻴﺜﻜﺗ .1 ،ﺯﺎﺘﻨﻣﺮﻓﺯﺎﺑﺖﻔﺟ 100 ﺮﻛﺭﺎﻣ،PCRﺕﻻﻮﺼﺤﻣ .B .2 ﻩﺭﺎﻤﺷ

ﻦﻴﻟﻮﺑﻮﺗﺎﺘﺑﻲﺻﺎﺼﺘﺧﺍﻱﺎﻫﺮﻤﻳﺍﺮﭘﺎﺑcDNAﺮﻴﺜﻜﺗ .4 ،ﻲﻔﻨﻣ

(5)

pEGFP-C1ﻲﻧﺎﻴﺑﺭﻮﺘﻛﻭﺭﺩPEP cDNAﻥﺩﺮﻛﻥﻮﻠﻛﺏﺎﺳ ﺭﻮﺘﻛﻭﻪﺑ PEP cDNAﻪﺑ ﻁﻮﺑﺮﻣPCRﻝﻮﺼﺤﻣﻝﺎﻘﺘﻧﺍﺯﺍ ﺲﭘ ﻯﻭﺎﺣﻂﻴﺤﻣﺭﺩﻲﻧﻮﻠﻛ 25 ،ﻰﻳﺎﻳﺮﺘﻛﺎﺑﻡﺭﻮﻓﺲﻧﺍﺮﺗﻭ pEGFP-C1ﻰﻧﺎﻴﺑ

.ﺪﻧﺩﺮﻛﺪﺷﺭﻚﻴﺗﻮﻴﺑﻰﺘﻧﺁ ﺖﺳﺩﻪﺑﻯﺎﻫﻰﻧﻮﻠﻛﺭﺩPEP cDNAﺩﻮﺟﻭﻰﻳﺎﻬﻧﺪﻴﻳﺎﺗﺖﻬﺟ pep-R Sal ﻭ pep-F Bgl II ﻯﺎﻫﺮﻤﻳﺍﺮﭘﺎﺑ PCRﺶﻨﻛﺍﻭ ﻩﺪﻣﺁ 8 ﺩﻮﺟﻭﺪﻳﻮﻣ ﻥﺁ ﺞﻳﺎﺘﻧ ﻪﻛ ﺖﻓﺮﮔﺕﺭﻮﺻ ﺎﻫﻰﻧﻮﻠﻛ ﻦﻳﺍ ﻯﺍﺮﺑ I ﺩﻮﺟﻭﺪﻴﻳﺎﺗﻪﺠﻴﺘﻧ 2 ﻞﻜﺷﺭﺩ .ﺩﻮﺑﺐﻴﻛﺮﺗﻮﻧﺪﻴﻤﺳﻼﭘﻯﻭﺎﺣﻲﻧﻮﻠﻛ ﻪﻧﻮﻤﻧﻥﺍﻮﻨﻋ ﻪﺑ ﻲﻧﻮﻠﻛ ﺶﺷﻥﺎﻴﻣ ﺯﺍ ﻲﻧﻮﻠﻛ ﻭﺩﺭﺩ PEP cDNA .ﺖﺳﺍﻩﺪﺷﻩﺩﺍﺩﻥﺎﺸﻧ

ﻲﻟﺍﻮﺗﻦﻴﻴﻌﺗ ﺪﻫﺩﻰﻣﻥﺎﺸﻧﺍﺭPEP cDNA ﻯﺯﺎﺑﺖﻔﺟ 630ﻰﻟﺍﻮﺗ ،3 ﻞﻜﺷ ﺖﻘﺑﺎﻄﻣﺶﻧﺍﺭﺎﻜﻤﻫ ﻭ ﺰﻨﻴﺗﺭﺎﻣﺭﺮﻓﻪﻟﺎﻘﻣﺭﺩﻩﺪﺷ ﺵﺭﺍﺰﮔﻰﻟﺍﻮﺗﺎﺑ ﻪﻛ ﻲﻟﺍﻮﺗ ﻥژ ﻞﻴﺴﻛﻮﺑﺮﻛ ﻱﺎﻬﺘﻧﺍ ﺭﺩ ﺖﺳﺍ ﺮﻛﺫ ﻪﺑ ﻡﺯﻻ .(16) ﺩﺭﺍﺩ ﻦﻴﺳﻮﻟﻭﺰﻳﺍ -ﻦﻳﺰﻴﻟ -ﻦﻳﺮﺳ ﻪﻨﻴﻣﺁ ﻱﺎﻫﺪﻴﺳﺍ ﻪﺑ ﻪﻛ AGCAAGATA ﺩﻮﺟﻭ،ﺖﺳﺍPTS1ﻩﺪﺷﻆﻔﺣﻲﻟﺍﻮﺗﻪﺑﻪﻴﺒﺷﻭﺩﻮﺷﻰﻣﻪﻤﺟﺮﺗ (SKI) ﻭ EGFP-C1 Fﻯﺎﻫﺮﻤﻳﺍﺮﭘﺎﺑﺐﻴﻛﺮﺗﻮﻧﺪﻴﻤﺳﻼﭘﻰﻟﺍﻮﺗﻦﻴﻴﻌﺗ .ﺩﺭﺍﺩ

ﻪﺑEGFP-C1ﺭﻮﺘﻛﻭﺭﺩ PEP cDNAﻪﻛﺩﺍﺩﻥﺎﺸﻧ EGFP-C1 R .ﺖﺳﺍﻪﺘﻓﺮﮔﺭﺍﺮﻗEGFPﻥژﺎﺑ ﺶﻧﺍﻮﺧﻢﻫﺕﺭﻮﺻ

ﺯﺍﻞﺻﺎﺣ ﻱﺎﻫﻲﻧﻮﻠﻛﺭﺩ PEP cDNA ﺩﻮﺟﻭﻲﺳﺭﺮﺑ ﺞﻳﺎﺘﻧ :2 ﻞﻜﺷ

ﺩﻮﺷﻲﻣﻪﻈﺣﻼﻣﻪﻛﺭﻮﻄﻧﺎﻤﻫ .PCRﺯﺍﻩﺩﺎﻔﺘﺳﺍﺎﺑﻰﻳﺎﻳﺮﺘﻛﺎﺑﻡﺭﻮﻓﺲﻧﺍﺮﺗ

ﻭﺩ 2 ﻭ 1 ﻱﺎﻫﻥﻮﺘﺳﻭPEP cDNAﺪﻗﺎﻓﻱﺎﻫﻲﻧﻮﻠﻛ 6 ﺎﺗ 3ﻱﺎﻫﻥﻮﺘﺳ

.ﺪﻨﺷﺎﺑﻲﻣPEP cDNAﻱﻭﺎﺣﻲﻧﻮﻠﻛ Ladder 1 2 3 4 5 6

1000

646 (bp)

ﻦﻳﺍﺭﺎﺘﺧﺎﺳﺭﺩﺖﺳﺍﻩﺪﺷﻩﺩﺍﺩﻥﺎﺸﻧﻞﻜﺷﺭﺩﻪﻛﺭﻮﻃﻥﺎﻤﻫ،SMARTﻪﻣﺎﻧﺮﺑﺎﺑPEP ﻦﻴﺌﺗﻭﺮﭘﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻑﺩﺍﺮﺗﺰﻴﻟﺎﻧﺁ (A :4 ﻞﻜﺷ

ﻪﻨﻣﺍﺩﻚﻳﻭ 169 ﺎﺗ 147 ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻪﻴﺣﺎﻧﺭﺩﻰﻳﺎﺸﻏﻥﻭﺭﺩﻪﻌﻄﻗﻚﻳ،28 ﺎﺗ 1 ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻪﻴﺣﺎﻧﺭﺩﺪﻴﺘﭙﭘﻝﺎﻨﮕﻴﺳﻚﻳﻦﻴﺌﺗﻭﺮﭘ

.ﺩﺭﺍﺩﺩﻮﺟﻭ 111 ﺎﺗ 31 ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻪﻴﺣﺎﻧﺭﺩ 3 ﻉﻮﻧﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓ

ﻩﺩﺍﺩﻥﺎﺸﻧﻞﻜﺷﺭﺩﻪﻛﺭﻮﻃﻥﺎﻤﻫ،Bio EditﺭﺍﺰﻓﺍﻡﺮﻧﺯﺍﻩﺩﺎﻔﺘﺳﺍﺎﺑPEPﻦﻴﺌﺗﻭﺮﭘﺭﺩﺩﻮﺟﻮﻣﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻱﺰﻳﺮﮔﺏﺁﻥﺍﺰﻴﻣﻰﺳﺭﺮﺑ (B

.ﺩﺭﺍﺩﺩﻮﺟﻭ 32 ﺎﺗ 12ﻭ 169 ﺎﺗ 152 ﻩﺭﺎﻤﺷﻱﺎﻫﻪﻨﻴﻣﺁﺪﻴﺳﺍﺯﺍﺰﻳﺮﮔﺏﺁﻩﺮﻴﺠﻧﺯﻭﺩﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻥﺎﻤﺘﺧﺎﺳﺭﺩﺖﺳﺍﻩﺪﺷ

ﺵﻮﻣPEP cDNAﮓﻨﻴﻧﻮﻠﻛ

A

1 100 200

ﻩﺪﺷﮓﻧﺭﺮﭘﺖﻤﺴﻗ .ﺪﺷﺎﺑﻲﻣPvu IIﻢﻳﺰﻧﺁﻂﺳﻮﺗﺖﺴﻜﺷﻱﺎﻫﻩﺎﮕﻳﺎﺟﺖﺳﺍﻩﺪﺷﺺﺨﺸﻣﻂﺧﺮﻳﺯﺎﺑﻪﻛﻰﻳﺎﻫﺪﻴﺗﻮﺌﻠﻛﻮﻧﻰﻟﺍﻮﺗ،PEP cDNAﻲﻟﺍﻮﺗ :3 ﻞﻜﺷ

ﮓﻧﺭﺮﭘﻂﺧﺮﻳﺯﺎﺑﻪﻛﻰﻳﺎﻫﻪﻨﻴﻣﺁﺪﻴﺳﺍﻰﻟﺍﻮﺗ .ﺖﺳﺍﺰﻳﺮﮔﺏﺁﻯﺎﻫﻩﺮﻴﺠﻧﺯﻪﺑﻁﻮﺑﺮﻣﻞﻴﻄﺘﺴﻣﺭﺩﻪﺘﻓﺮﮔﺭﺍﺮﻗﻯﺎﻫﻪﻨﻴﻣﺁﺪﻴﺳﺍﻰﻟﺍﻮﺗ .ﺖﺳﺍSKIﻲﻟﺍﻮﺗﻰﻳﺎﻬﺘﻧﺍ

.ﺖﺳﺍ 3 ﻉﻮﻧﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓﻪﻨﻣﺍﺩ،ﻩﺪﻳﺩﺮﮔﺺﺨﺸﻣ

B

---

---Mean Hydrophobicity

2.7 1.8 0.9

-0.9 -1.8 -2.7 0

-3.6

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 Position

(6)

PEPﻦﻴﺌﺗﻭﺮﭘﺭﺎﺘﺧﺎﺳﻲﺳﺭﺮﺑ ﻱﺎﻫﻪﻣﺎﻧﺮﺑ ﺯﺍ PEP ﻦﻴﺌﺗﻭﺮﭘ ﻲﻟﺎﻤﺘﺣﺍ ﺭﺎﺘﺧﺎﺳ ﻲﺳﺭﺮﺑ ﻱﺍﺮﺑ ﻪﻣﺎﻧﺮﺑ ﺎﺑ PEP ﺰﻴﻟﺎﻧﺁ ﺞﻳﺎﺘﻧ .ﺪﺷ ﻩﺩﺎﻔﺘﺳﺍ ﻲﻜﻴﺗﺎﻣﺭﻮﻔﻧﺍﻮﻴﺑ ﻒﻠﺘﺨﻣ ﺲﻜﻴﻠﻫﺎﻔﻟﺁﺭﺎﺘﺧﺎﺳﻭﺩ ﺩﻮﺟﻭﻱﻻﺎﺑﻝﺎﻤﺘﺣﺍﻩﺪﻨﻫﺩﻥﺎﺸﻧ TMpred 170 ﺎﺗ 152 ﻭ 31 ﺎﺗ 12 ﻱﺎﻫﻪﻨﻴﻣﺁﺪﻴﺳﺍ ﻦﻴﺑ ﻪﻴﺣﺎﻧ ﻭﺩ ﺭﺩ ﻲﻳﺎﺸﻏ ﺯﺍ ﻻﺎﺑ ﻪﻌﻟﺎﻄﻣ ﺯﺍ ﻞﺻﺎﺣ ﺞﻳﺎﺘﻧ ﺪﻴﻳﺎﺗ ﺭﻮﻈﻨﻣ ﻪﺑ ﻦﻴﻨﭼﻢﻫ .ﺪﺷﺎﺑﻲﻣ ﻒﻠﺘﺨﻣﻱﺎﻫﻪﻨﻣﺍﺩﻥﺍﻮﺗﻲﻣﻥﺁﺎﺑﻪﻛ SMARTﻡﺎﻧﻪﺑﻱﺮﮕﻳﺩﻪﻣﺎﻧﺮﺑ ﻱﺎﻫﺪﻴﺳﺍ ﻑﺩﺍﺮﺗ ﺰﻴﻟﺎﻧﺁ .ﺪﻳﺩﺮﮔﻩﺩﺎﻔﺘﺳﺍ ،ﺩﻮﻤﻧﻲﻳﺎﺳﺎﻨﺷ ﺍﺭﻦﻴﺌﺗﻭﺮﭘ ﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﺭﺎﺘﺧﺎﺳﺭﺩﻪﻛﺩﺍﺩﻥﺎﺸﻧﻪﻣﺎﻧﺮﺑﻦﻳﺍﺎﺑPEPﻦﻴﺌﺗﻭﺮﭘﻪﻨﻴﻣﺁ ﻥﻭﺭﺩﻪﻌﻄﻗﻚﻳ،28 ﺎﺗ 1 ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻪﻴﺣﺎﻧﺭﺩﺪﻴﺘﭙﭘﻝﺎﻨﮕﻴﺳﻚﻳ ﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓﻪﻨﻣﺍﺩﻚﻳﻭ 169 ﺎﺗ 147 ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻪﻴﺣﺎﻧﺭﺩﻰﻳﺎﺸﻏ .(4A ﻞﻜﺷ) ﺩﺭﺍﺩﺩﻮﺟﻭ 111 ﺎﺗ 31 ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍﻪﻴﺣﺎﻧﺭﺩ 3 ﻉﻮﻧ ﻪﻨﻴﻣﺁﻱﺎﻫﺪﻴﺳﺍ ﻱﺰﻳﺮﮔﺏﺁ ﻥﺍﺰﻴﻣBio Edit ﺭﺍﺰﻓﺍﻡﺮﻧﺯﺍ ﻩﺩﺎﻔﺘﺳﺍﺎﺑ ﺶﻧﺍﻮﺧﺎﺑKyte and DoolittleﺵﻭﺭﺎﺑPEPﻦﻴﺌﺗﻭﺮﭘﺭﺩﺩﻮﺟﻮﻣ ﺭﺩﻪﻛﺪﻳﺩﺮﮔﺺﺨﺸﻣﻭﺖﻓﺮﮔﻡﺎﺠﻧﺍﻩﺮﺠﻨﭘﺮﻫﺭﺩﻪﻨﻴﻣﺁﺪﻴﺳﺍ 13 ﻩﺭﺎﻤﺷ ﻱﺎﻫﻪﻨﻴﻣﺁﺪﻴﺳﺍ ﺯﺍ ﺰﻳﺮﮕﺑﺁ ﻩﺮﻴﺠﻧﺯ ﻭﺩﻦﻴﺌﺗﻭﺮﭘ ﻦﻳﺍ ﻥﺎﻤﺘﺧﺎﺳ .(4Bﻞﻜﺷ) ﺩﺭﺍﺩﺩﻮﺟﻭ 32 ﺎﺗ 12ﻭ 169 ﺎﺗ 152

PEPﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﻦﻴﺌﺗﻭﺮﭘﻲﺑﺎﻳﺩﺭ ﻝﺎﻨﮕﻴﺳ PEP ﻦﻴﺌﺗﻭﺮﭘ ﻞﻴﺴﻛﻮﺑﺮﻛ ﻱﺎﻬﺘﻧﺍ ﺭﺩ ﻪﻛ ﺎﺠﻧﺁ ﺯﺍ ﻪﺑ ﻦﻴﺌﺗﻭﺮﭘ ﻦﻳﺍ ﺩﻭﺭﻲﻣ ﺭﺎﻈﺘﻧﺍ ،ﺩﺭﺍﺩ ﺩﻮﺟﻭ PTS1 ﻪﺑﺎﺸﻣ ﻪﻧﺎﺸﻧ ﻝﺎﺒﻧﺩ ﻪﺑ PEP cDNA ﻦﺘﻓﺮﮔ ﺭﺍﺮﻗ ﺎﺑ ﻭ ﺩﻮﺷ ﺩﺭﺍﻭ ﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻥﺎﻤﻫ .ﺩﺮﻛﻲﺑﺎﻳﺩﺭﺍﺭﻥﺁﻥﺍﻮﺘﺑ،ﻲﺸﻧﺍﻮﺧﺐﻟﺎﻗﻚﻳﺭﺩEGFPﻥژ EGFP ﺖﻨﺳﺭﻮﻠﻓ ﺰﺒﺳ ﮓﻧﺭ ،ﺪﺷ ﻩﺩﺍﺩ ﻥﺎﺸﻧ 5 ﻞﻜﺷ ﺭﺩﻪﻛﺭﻮﻃ ﺐﻴﻛﺮﺗﻮﻧ ﺪﻴﻤﺳﻼﭘ ﻥﺪﺷ ﺩﺭﺍﻭ ﺮﮕﻧﺎﻴﺑ CHO ﻝﻮﻠﺳ ﺯﺍ ﻰﺘﻴﻌﻤﺟ ﺭﺩ

ﺪﺷﺎﺑﻰﻣﺭﻮﻛﺬﻣﻯﺎﻫﻝﻮﻠﺳﺭﺩﺪﻴﻤﺳﻼﭘﻥﺎﻴﺑﻭﺎﻫﻝﻮﻠﺳﻦﻳﺍﻥﻭﺭﺩ ﻪﺑ ﻁﻮﺑﺮﻣ ﺖﻨﺳﺭﻮﻠﻓ ﻲﮔﺪﻨﻛﺍﺮﭘ ﻯﻮﮕﻟﺍ ﻪﺑﺎﺸﺗ ﻦﻳﺍ ﺮﺑ ﻩﻭﻼﻋ .(C،A) ﻦﻳﺍ ﻥﺪﺷ ﺩﺭﺍﻭ ﺮﮕﻧﺎﻴﺑ ،(D،B) ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﺯﻻﺎﺗﺎﻛﺎﺑ EGFP ﻪﺑ .ﺪﺷﺎﺑﻰﻣﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻥﻭﺭﺩﻪﺑﻦﻴﺌﺗﻭﺮﭘ

ﺚﺤﺑ

ﻰﻳﺎﺳﺎﻨﺷﻪﺑﻲﺴﻜﻴﻣﻮﺌﺗﻭﺮﭘﻭﻲﻜﻴﺘﻧژﻱﺎﻫﺵﻭﺭﺯﺍﻩﺩﺎﻔﺘﺳﺍﻥﻮﻨﻛﺎﺗ ،ﺪﺷﺎﺑﻰﻣ ﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﺰﻧژﻮﻴﺑ ﻪﻣﺯﻻ ﻪﻛ ﻦﻴﺴﻛﺍﺮﭘ ﻦﻴﺌﺗﻭﺮﭘ ﻉﻮﻧ 32 ﻡﻭﺰﻴﺴﻛﺍﺮﭘﻥﺪﺷﻢﻴﺴﻘﺗﻝﻮﺌﺴﻣﺎﻫﻦﻴﺴﻛﺍﺮﭘﺯﺍﻲﻀﻌﺑ .ﺖﺳﺍﻩﺪﺷﺮﺠﻨﻣ ﺪﻨﺷﺎﺑﻲﻣ ﻲﻠﺒﻗﻱﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﺯﺍﺮﺗﺪﻳﺪﺟﻱﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘﻞﻴﻜﺸﺗ ﻭ ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﺲﻜﻳﺮﺗﺎﻣﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘﺖﻳﺍﺪﻫﻭﻝﺎﻘﺘﻧﺍﺭﺩﺎﻬﻧﺁﺮﺘﺸﻴﺑﺎﻣﺍ ﺖﻠﻋ ﻪﺑ ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘﻱﺎﻫﻱﺭﺎﻤﻴﺑ ﺯﺍ ﻱﺭﺎﻴﺴﺑ .(17 ،6) ﺪﻧﺭﺍﺩ ﺶﻘﻧ ﻥﻭﺭﺩ ﻪﺑ ﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﺲﻜﻳﺮﺗﺎﻣ ﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘ ﻝﺎﻘﺘﻧﺍ ﺭﺩ ﺺﻘﻧ ﻞﻧﺎﮔﺭﺍﻦﻳﺍﺭﺩﻊﻗﺍﻭﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘﺩﺍﺪﻌﺗﻪﭼﺮﻫﻭﺪﻨﺷﺎﺑﻲﻣﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻱﺎﻫﻱﺭﺎﻤﻴﺑ ﻭ ﺎﻬﻧﺁ ﻦﻴﺑ ﻱﺮﺘﺸﻴﺑ ﻁﺎﺒﺗﺭﺍ ﻻﺎﻤﺘﺣﺍ ﺩﻮﺷ ﻪﺘﺧﺎﻨﺷ ﺮﺘﺸﻴﺑ .ﺩﻮﺷﻲﻣﺖﻓﺎﻳﻲﻣﻭﺰﻴﺴﻛﺍﺮﭘءﺎﺸﻨﻣﺎﺑ ﻲﻧﺎﺴﻧﺍ ﺮﺛﺆﻣ ﺵﻭﺭﻚﻳﻲﻧﺎﻴﺑﺭﻮﺘﻛﻭ ﺯﺍ ﻩﺩﺎﻔﺘﺳﺍﺎﺑ ﮓﻨﻴﻧﻮﻠﻛﻱﮋﺗﺍﺮﺘﺳﺍ ﺮﻈﻧﻪﺑﻪﻛﺎﺠﻧﺁﺯﺍ .(18) ﺪﺷﺎﺑﻲﻣﻒﻠﺘﺨﻣﻱﺎﻫﻥژﺖﻴﻟﺎﻌﻓﻙﺭﺩﺭﺩ ﻰﻤﻬﻣ ﺶﻘﻧ ﻦﻴﻨﺟ ﻞﻣﺎﻜﺗ ﻭ ﺪﺷﺭ ﻰﻃ ﺭﺩ PEP ﻦﻴﺌﺗﻭﺮﭘ ﺪﺳﺭﻰﻣ ﻢﻳﺪﺷﻥﺁ ﺮﺑﻪﻨﻴﻣﺯ ﻦﻳﺍ ﺭﺩﺮﺘﺸﻴﺑﺕﺎﻌﻟﺎﻄﻣ ﺖﻬﺟ،(16) ﺪﺷﺎﺑ ﻪﺘﺷﺍﺩ cDNA ﻪﻌﻟﺎﻄﻣ ﻦﻳﺍ ﺭﺩ .ﻢﻴﻳﺎﻤﻧ ﻥﻮﻠﻛ ﺍﺭ ﻥژ ﻦﻳﺍ cDNA ﺍﺪﺘﺑﺍ ﻪﻛ ﺭﺩﻥﺁ ﻥﺎﻴﺑ ﻝﺎﺒﻧﺩﻪﺑ ﻭ ﻥﻮﻠﻛpEGFP-C1 ﻲﻧﺎﻴﺑﺭﻮﺘﻛﻭ ﺭﺩPEP ﻱﺮﻴﮔﺭﺍﺮﻗ ﻞﺤﻣ ﻥﻮﭽﻤﻫ ﻰﻔﻠﺘﺨﻣ ﻱﺎﻫﻪﺒﻨﺟ ﻥﺍﺭﺍﺪﻧﺎﺘﺴﭘ ﻱﺎﻫﻝﻮﻠﺳ .ﺖﻓﺮﮔﺭﺍﺮﻗﻲﺳﺭﺮﺑﺩﺭﻮﻣﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻰﻠﻧﺎﮔﺭﺍﻥﻭﺭﺩ

ﺰﺒﺳﻁﺎﻘﻧ .ﺯﻻﺎﺗﺎﻛﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘﻯﺩﺎﺑﻰﺘﻧﺁﺎﺑﻯﺰﻴﻣﺁﮓﻧﺭﻭEGFP-PEPﺪﻴﻤﺳﻼﭘﺎﺑﺎﻫﻝﻮﻠﺳﺖﻜﻓﺲﻧﺍﺮﺗ :5 ﻞﻜﺷ

ﻯﺰﻴﻣﺁﮓﻧﺭﺎﻫﻝﻮﻠﺳﻥﻭﺭﺩﻩﺪﻨﻛﺍﺮﭘﮓﻧﺭﺰﻣﺮﻗﻁﺎﻘﻧ،(A،B) EGFP-PEPﻦﻴﺌﺗﻭﺮﭘﻥﺎﻴﺑ،ﺎﻫﻝﻮﻠﺳﻥﻭﺭﺩﻩﺪﻨﻛﺍﺮﭘﮓﻧﺭ

ﻯﻮﮕﻟﺍ .(D،C) ﺪﻫﺩﻰﻣﻥﺎﺸﻧﺍﺭTexas Redﻪﻳﻮﻧﺎﺛﻯﺩﺎﺑﻰﺘﻧﺁﻭﺯﻻﺎﺗﺎﻛﻰﺻﺎﺼﺘﺧﺍﻪﻴﻟﻭﺍﻯﺩﺎﺑﻰﺘﻧﺁﺎﺑﺯﻻﺎﺗﺎﻛﻢﻳﺰﻧﺁ

.ﺖﺳﺍﻝﻮﻣﻭﺮﻜﻴﻣ 20 ﺎﺑﺮﺑﺍﺮﺑﺭﺎﺑ .ﺪﺷﺎﺑﻰﻣPEPﻥﺩﻮﺑﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘﺮﮕﻧﺎﻴﺑﮓﻧﺭﻭﺩﻦﻳﺍﻪﺑﺎﺸﻣﻰﻧﺎﻜﻣ

A

B

(7)

ﺐﻠﻗ ﺖﻓﺎﺑﺯﺍ PEP cDNA ﮓﻨﻴﻧﻮﻠﻛ ﻱﺍﺮﺑ RNA ﺝﺍﺮﺨﺘﺳﺍ ﺖﻓﺎﺑ ﺭﺩ ﻪﻛ ﺪﻫﺩﻲﻣ ﻥﺎﺸﻧ ﻲﻠﺒﻗ ﺕﺎﻌﻟﺎﻄﻣ .ﺖﻓﺮﮔ ﺕﺭﻮﺻ ﺵﻮﻣ ﺮﻳﺎﺳ ﺯﺍ ﺰﻐﻣ ﻭ ﻲﺘﻠﻜﺳﺍ ﻪﻠﻀﻋ ﺖﻓﺎﺑ ﺪﻨﻧﺎﻤﻫ ﻥژ ﻦﻳﺍ ﻥﺎﻴﺑ ،ﺐﻠﻗ cDNA ﺪﻴﻳﺎﺗ ﻲﺳﺭﺮﺑ ﺭﻮﻈﻨﻣ ﻪﺑ .(16) ﺖﺳﺍ ﺮﺘﺸﻴﺑ ﺮﮕﻳﺩ ﻱﺎﻫﺖﻓﺎﺑ β-tubulin 5ﻥژ ﺯﺍﻱﺍﻪﻌﻄﻗﻯﺍﺮﺑ RT-PCR ﺶﻨﻛﺍﻭﻩﺪﺷﺰﺘﻨﺳ ﺎﻫﺖﻓﺎﺑﻡﺎﻤﺗﺭﺩﻩﺪﺷﻥﺎﻴﺑ Housekeeping Geneﻚﻳﻥﺍﻮﻨﻋﻪﺑ ﻪﻌﻄﻗﻲﻟﺍﻮﺗﻦﻴﻴﻌﺗﺖﻳﺎﻬﻧﺭﺩﻭPvu IIﻢﻳﺰﻧﺁﺎﺑﺵﺮﺑ .(19) ﺪﺷﻡﺎﺠﻧﺍ PEP cDNAﻥﺎﻤﻫﻩﺪﺷﻥﻮﻠﻛcDNA ﻪﻛﺩﻮﻤﻧﺖﺑﺎﺛﻩﺪﺷﻥﻮﻠﻛ ﻪﻛ ﺪﻫﺩﻲﻣ ﻥﺎﺸﻧ cDNA ﻦﻳﺍ ﻱﺎﻫﺯﺎﺑ ﻑﺩﺍﺮﺗ ﻲﺳﺭﺮﺑ .ﺪﺷﺎﺑﻲﻣ

ﺯﺎﺑﺖﻔﺟ 630 ﺎﺑ PEP ﻥژ Open Reading Frame (ORF)

ﺎﺑ ﻚﻴﺗﺎﻣﺭﻮﻔﻧﺍﻮﻴﺑ ﺕﺎﻌﻟﺎﻄﻣ .ﺪﺷﺎﺑﻲﻣ ﻪﻨﻴﻣﺁﺪﻴﺳﺍ 209 ﻩﺪﻨﻨﻛﺪﻛ ﻪﻨﻣﺍﺩﻚﻳﻱﺍﺭﺍﺩPEPﻦﻴﺌﺗﻭﺮﭘﻪﻛﺩﺍﺩﻥﺎﺸﻧﻒﻠﺘﺨﻣﻱﺎﻫﺭﺍﺰﻓﺍﻡﺮﻧ ﻪﻛ (114 ﺎﺗ 31 ﻪﻨﻴﻣﺁﺪﻴﺳﺍﺯﺍ) ﺪﺷﺎﺑﻲﻣ 3 ﻉﻮﻧﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓﺎﺑﻪﺑﺎﺸﻣ ﺮﻈﻧ ﺩﺭﻮﻣ ﻦﻴﺌﺗﻭﺮﭘ ﺭﺩ ﻪﻨﻣﺍﺩ ﻦﻳﺍ ﻲﻟﺎﻤﺘﺣﺍ ﺶﻘﻧ ﺩﺭﻮﻣ ﺭﺩ ﻥﻮﻨﻛﺎﺗ ﻦﻴﺘﻜﻧﻭﺮﺒﻴﻓﻪﻨﻣﺍﺩﺪﺟﺍﻭﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘ .ﺖﺳﺍﻩﺪﻣﺎﻴﻧﺖﺳﺩﻪﺑﻲﺗﺎﻋﻼﻃﺍ ﻲﺗﻮﻳﺭﺎﻛﻭﺮﭘﻭﻲﺗﻮﻳﺭﺎﻛﻮﻳﻱﺎﻫﻝﻮﻠﺳﺭﺩﻲﻔﻠﺘﺨﻣﻱﺎﻫﺖﻴﻟﺎﻌﻓﻱﺍﺭﺍﺩ ﺕﻻﺎﺼﺗﺍﺭﺩﺎﻫﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﺶﻘﻧﻪﺑﻥﺍﻮﺗﻲﻣﻪﻠﻤﺟﻥﺁﺯﺍﻪﻛﺪﻨﺷﺎﺑﻲﻣ ﻲﻟﻮﻠﺳﺖﻠﻜﺳﺍﻥﺎﻤﺘﺧﺎﺳﺭﺩﻦﻴﻨﭼﻢﻫﻭﻰﻳﺎﺸﻏﻱﺎﻫﻩﺪﻧﺮﻴﮔ،ﻲﻟﻮﻠﺳ ﻥﺎﻤﺘﺧﺎﺳ ﺭﺩ ﻪﻨﻣﺍﺩ ﻦﻳﺍﺭﻮﻀﺣ ﻦﻳﺍﺮﺑﺎﻨﺑ .(20-23) ﺩﻮﻤﻧ ﻩﺭﺎﺷﺍ ﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻲﻟﺎﻤﺘﺣﺍ ﺶﻨﻛﻢﻫﺮﺑﺩﻮﺟﻭ ﻩﺪﻨﻫﺩﻥﺎﺸﻧPEPﻦﻴﺌﺗﻭﺮﭘ ﺶﻘﻧ ﻪﺑ ﻪﺟﻮﺗ ﺎﺑ ﻩﺪﻳﺍ ﻦﻳﺍ .ﺪﺷﺎﺑﻲﻣ ﻪﺘﺧﺎﻨﺷﺎﻧ ﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘ ﺮﮕﻳﺩ ﺎﺑ ﻲﻟﻮﻠﺳﻞﺧﺍﺩﻰﮔﺪﻨﻛﺍﺮﭘﻭﻡﻭﺰﻴﺴﻛﺍﺮﭘﺰﻧژﻮﻓﺭﻮﻣﺭﺩﻲﻟﻮﻠﺳﺖﻠﻜﺳﺍ ﻲﺳﺭﺮﺑ ﻭ ﺮﺘﺸﻴﺑ ﺕﺎﻌﻟﺎﻄﻣ ﺍﺬﻟ .(24) ﺩﺩﺮﮔﻲﻣ ﺖﻳﻮﻘﺗ ﻞﻧﺎﮔﺭﺍ ﻦﻳﺍ .ﺪﺳﺭﻰﻣﺮﻈﻧﻪﺑﻱﺭﻭﺮﺿﺩﺭﻮﻣﻦﻳﺍﺭﺩPEP ﻦﻴﺌﺗﻭﺮﭘﻲﻟﺎﻤﺘﺣﺍﺶﻘﻧ ﻭﺩﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻥﺎﻤﺘﺧﺎﺳﺭﺩﻪﻛﺩﺍﺩﻥﺎﺸﻧﻚﻴﺗﺎﻣﺭﻮﻔﻧﺍﻮﻴﺑﺕﺎﻌﻟﺎﻄﻣ (169 ﺎﺗ 159 ﻭ 32 ﺎﺗ 12 ﻩﺭﺎﻤﺷ ﻪﻨﻴﻣﺁﻯﺎﻫﺪﻴﺳﺍﺯﺍ) ﺰﻳﺮﮔﺏﺁﻪﻨﻣﺍﺩ ﺎﻔﻟﺁ ﺭﺎﺘﺧﺎﺳﻱﺍﺭﺍﺩﺩﺎﻳﺯ ﻝﺎﻤﺘﺣﺍ ﻪﺑﻪﻛ ﻪﻨﻣﺍﺩﻭﺩﻦﻳﺍ .ﺩﻮﺷﻲﻣﻩﺪﻳﺩ

ﺭﺩ ﻲﺿﺮﻓﻰﻳﺎﺸﻏ ﻥﻭﺭﺩ ﻱﺎﻫﻪﻨﻣﺍﺩﺩﻮﺟﻭﺮﮕﻧﺎﻴﺑ ،ﺪﻨﺷﺎﺑﻲﻣﺲﻜﻴﻠﻫ ﻱﻭﺭﺮﺑﻱﺮﺘﺸﻴﺑﺕﺎﻌﻟﺎﻄﻣ ﺪﻳﺎﺑﻲﻣﻪﺠﻴﺘﻧﺭﺩ .ﺖﺳﺍﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻞﺧﺍﺩ ﺭﺩﻦﻴﺌﺗﻭﺮﭘﻦﻳﺍﻩﻭﻼﻋﻪﺑ .ﺩﺮﻴﮕﺑﺕﺭﻮﺻPEP ﻦﻴﺌﺗﻭﺮﭘﻥﺩﻮﺑﻲﻳﺎﺸﻏ ﺖﻳﺍﺪﻫﻝﻮﺌﺴﻣﻪﻛ- SKLﺪﻴﺘﭙﭘﻪﺑﺎﺸﻣSKIﺪﻴﺘﭙﭘﻯﺍﺭﺍﺩﺩﻮﺧﻱﺎﻬﺘﻧﺍ .ﺪﺷﺎﺑﻲﻣ - ﺖﺳﺍﻡﻭﺰﻴﺴﻛﺍﺮﭘﻞﺧﺍﺩﻪﺑﻰﺴﻜﻳﺮﺗﺎﻣﻱﺎﻫﻦﻴﺌﺗﻭﺮﭘ

ﻯﺮﻴﮔ

ﻪﺠﻴﺘﻧ

C2C12ﻱﺎﻫﻝﻮﻠﺳﻯﻭﺭﺰﻨﻴﺗﺭﺎﻣﺮﻳﺮﻓﺕﺎﻌﻟﺎﻄﻣﺯﺍﻞﺻﺎﺣﺞﻳﺎﺘﻧ ﻞﺼﺘﻣPEP ﻦﻴﺌﺗﻭﺮﭘﻪﻛﺩﺍﺩ ﻥﺎﺸﻧ HepG2ﻭ Ala-T1 ﻭ 293 ﻭ

ﺭﺩ .(16) ﺩﺩﺮﮔﻲﻣ ﺩﺭﺍﻭ ﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻥﻭﺭﺩ ﻪﺑ EGFP ﻪﺑ ﻩﺪﺷ

ﻥﻭﺭﺩEGFPﻪﺑﻞﺼﺘﻣPEP ﻦﻴﺌﺗﻭﺮﭘﻱﺮﻴﮔﻯﺎﺟﺰﻴﻧﺮﺿﺎﺣﻪﻌﻟﺎﻄﻣ ﺕﺎﻌﻟﺎﻄﻣ ﻪﺘﺒﻟﺍ .ﺪﻳﺩﺮﮔ ﺪﻴﻳﺎﺗ CHO ﻝﻮﻠﺳ ﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﻚﻣﺍﺪﻧﺍ ﻥﻭﺭﺩ ﻯﺮﻴﮔﻯﺎﺟ ﻲﺳﺭﺮﺑ ﻭ SKI ﺪﻴﺘﭙﭘ ﻑﺬﺣ ﺯﺍ ﻩﺩﺎﻔﺘﺳﺍ ﺎﺑ ﻩﺪﻨﻳﺁ ﺪﻴﻳﺎﺗ ﻲﻠﻣﺎﻛ ﺕﺭﻮﺻ ﻪﺑ ﺪﻧﺍﻮﺗﻲﻣ SKI ﺪﻗﺎﻓ PEP ﻦﻴﺌﺗﻭﺮﭘ ﻲﻟﻮﻠﺳ ﻥﻭﺭﺩ ﻪﺑ PEP ﻦﻴﺌﺗﻭﺮﭘ ﻝﺎﻘﺘﻧﺍ ﺭﺩﻲﺘﻳﺍﺪﻫ ﻝﺎﻨﮕﻴﺳ ﻦﻳﺍ ﺶﻘﻧ ﻩﺪﻨﻨﻛ .ﺪﺷﺎﺑ ﺎﻫﻡﻭﺰﻴﺴﻛﺍﺮﭘ

ﺮﻜﺸﺗ

ﺮﻳﺪﻘﺗ

ﺏﻮﺼﻣ ﺡﺮﻃ ﺯﺍ ﻩژﻭﺮﭘ ﻦﻳﺍ ﻡﺎﺠﻧﺍ ﻯﺎﻫﻪﻨﻳﺰﻫ ﻩﺪﻤﻋ ﺶﺨﺑ ﻯﺍﺮﺑ ﻡﺯﻻﻰﻜﻴﺘﻧژﻯﺎﻫﺭﻮﺘﻛﺎﻓﻰﺳﺭﺮﺑ"ﻥﺍﻮﻨﻋ ﺎﺑﻥﺎﻳﻭﺭ ﻩﺪﻜﺸﻫﻭﮋﭘ

ﺶﺨﺑﻭ 85/5/10 ﺦﻳﺭﺎﺗﻪﺑ "ﻰﺒﺼﻋﻯﺎﻫﻝﻮﻠﺳﺰﻳﺎﻤﺗﺭﺩﻡﻭﺰﻴﺴﻛﺍﺮﭘ ﻭ ﮓﻨﻴﻧﻮﻠﻛ" ﻥﺍﻮﻨﻋ ﺎﺑ ﻥﺎﻬﻔﺻﺍ ﻩﺎﮕﺸﻧﺍﺩ ﺏﻮﺼﻣ ﺡﺮﻃ ﺯﺍ ﺮﮕﻳﺩ "ﺵﻮﻣ ﻰﻣﻭﺰﻴﺴﻛﺍﺮﭘ ﻦﻴﺌﺗﻭﺮﭘﻥژ cDNA ﺭﺎﺘﺧﺎﺳ ﺩﺮﻜﻠﻤﻋﻰﺑﺎﻳﺯﺭﺍ .ﺪﻳﺩﺮﮔﻦﻴﻣﺎﺗ 85/10/17 ﺦﻳﺭﺎﺗﻪﺑ 851017 ﻩﺭﺎﻤﺷﻪﺑ

References

1. Ferrer I, Kapfhammer J.P, Hindelang C, Kemp S, Troffer-Charlier N, Broccoli V, et al. Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmic reticulum damage. Hum Mol Genet. 2005; 14(23): 3565-3577.

2. Furuki S ,Tamura S ,Matsumoto N ,Miyata N, Moser A, Moser HW, et al. Mutations in the peroxin Pex26p responsible for peroxisome biogenesis disorders of complementation group 8 impair its stability, peroxi-somal localization, and interaction with Pex1p-Pex6p complex. J Biol Chem. 2006; 281(3): 1317-1323. 3. Sheikh FG, Pahan K, Khan M, Barbosa E, Singh I. Abnormality in catalase import into peroxisomes leads to severe neurological disorder. Proc Natl Acad Sci USA. 1998; 95: 2961-2966.

4. Shimizu N, Itoh R, Hirono Y, Otera H, Ghaedi K, Tateishi K, et al. The peroxin Pex14p: cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis. J Biol Chem. 1999; 274(18): 12593-12604. 5. Singha I, Paintliaa AS, Khana M, Stanislausa R, Paintliaa MK, Haqa E, et al. Impaired peroxisomal function in the central nervous system with inflamma-tory disease of experimental autoimmune encepha-lomyelitis animals and protection by lovastatin treat-ment. Brain Res. 2004; 1022(1-2): 1-11.

6. Brown LA, Baker A. Peroxisome biogenesis and the role of protein import. J Cell Mol Med. 2003; 7(4):388-400.

7. Ghaedi K, Tamura S, Okumoto K, Matsuzono Y, Fu-jiki Y. The peroxin Pex3p initiates membrane assem-bly in peroxisome biogenesis. Mol Biol Cell. 2000; 11(6): 2085-2102.

8. Michelakakis HM, Zafeiriou DI, Moraitou MS, Gootjes J, Wanders RJ. PEX1 deficiency presenting as leber congenital amaurosis. Pediatr Neurol. 2004; 31(2): 146-149.

9. Ofman1 R, Speijer D, Leen R ,Wanders R JA .Pro-teomic analysis of mouse kidney peroxisomes: iden-tification of RP2p as a peroxisomal nudix hydrolase with acyl-coenzyme A diphosphatase activity. Bio-chem J. 2006; 393(2): 537-543.

10. Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Ghaedi K, Fujiki Y, Kondo N. Identification of PEX3 as the gene mutated in a zellweger syndrome patient lacking peroxisomal remnant structures. Hum Mol Genet. 2000; 19(13): 1995-1999.

11. Baker A, Charlton W, Johnson B, Lopez-Huertas E, Oh J, Sparkes I, et al. Biochemical and molecular approaches to understanding protein import into per-oxisomes. Biochem Soc Trans. 2000; 28(4):499-504. 12. Baker A, Sparkes IA. Peroxisome protein import: some answers, more questions. Curr Opin Plant Biol. 2005; 8(6): 640-647.

13. Elgersma Y, Vos A, van den Berg M, Van Roer-mund C.W, Van der Sluijs P, Distel B, et al. Analysis of the carboxyl terminal peroxisomal targeting signal 1 in a homologous context in Saccharomyces cerevi-siae. J Biol Chem. 1996; 271(42): 26375-2682.

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14. Kamigaki A, Mano S, Terauchi K, Nishi Y, Tachibe Kinoshita Y, Nito K, et al. Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor. Plant J. 2003; 33(1): 161-175.

15. Mukai S, Ghaedi K, Fujiki Y. Intracellular locali-zation, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in mammalian cells. J Biol Chem. 2002; 277(11): 9548-9561.

16. Ferrer-Martínez A, Ruiz-Lozano P, Chien KR. Mouse PeP: A novel peroxisomal protein linked to myoblast differentiation and development. Dev Dyn. 2002; 224(2): 154-167.

17. Heiland I, Erdmann R. Biogenesis of peroxisomes: topogenesis of the peroxisomal membrane and matrix proteins. FEBS J. 2005; 272(10): 2362-2372.

18. Nyborg AC, Ladd TB, Jansen K, Kukar T, Golde TE. Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal pep-tide peptidase. FASEB J. 2006; 20(10): 1671-1679. 19. Bagutti C, Wobus A.M, Fässler R, Watt F.M. Differ-entiation of embryonal stem cells into keratinocytes:

comparison of wild-type and beta 1 integrin-deficient cells. Dev Biol. 1996; 179(1): 184-96.

20. Amodeo P, Fraternali F, Lesk AM, Pastore A. Modularity and homology: modelling of the titin type I modules and their interfaces. J Mol Biol. 2001; 311: 283-296.

21. Huang H, Sylvan J, Jonas M, Barresi R, So PT, Campbell KP, Lee RT. Cell stiffness and receptors: evidence for cytoskeletal subnetworks. Am J Physiol Cell Physiol. 2005; 288(1): 72-80.

22. Li L, Huang HH, Badilla CL, Fernandez JM. Me-chanical unfolding intermediates observed by single-molecule force spectroscopy in a fibronectin type III module. J Mol Biol, 2005; 345(4):816-826.

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