نژ ﮓﻨﻴﻧﻮﻠﻛ
p4
روژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ
* لددﺎﺷ ﻮﻨﻴﻣ ﺮﺘﻛد I يدﺰﻣروا دﺰﻣﺮﻫ ﺮﺘﻛد II ﻲﻗﻼﺧا ﻊﻣﻻ ﺮﺘﻛد III ﻲﻤﻇﺎﻛ ماﺮﻬﺑ ﺮﺘﻛد IV هﺪﻨﺑ نﺎﮔﮋﻣ رﻮﭘ V
هﺪﻴﻜﭼ
فﺪﻫ و ﻪﻨﻴﻣز : نژ p4 ﻲﻣ نﺎﻴﺑ ﻞﮕﻧا تﻮﮕﻴﺘﺳﺎﻣآ ﻪﻠﺣﺮﻣ رد ،روژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ ﺖـﻬﺟ ﺐـﺳﺎﻨﻣ ﺐﻴﻛﺮﺗ ًﻻﺎﻤﺘﺣا ﻪﻛ دﻮﺷ ﺑ ﻦﺴﻛاو ﻪﻴﻬﺗ ﺖﺳا زﻮﻴﻧﺎﻤﺸﻴﻟ ﻪﻴﻠﻋ ﺮ . رﻮـﺘﻛو ﻚـﻳ رد نژ ﻦﻳا نﻮﻠﻛ ،ﺶﻫوﮋﭘ ﻦﻳا مﺎﺠﻧا زا فﺪﻫ ) ﻞـﻗﺎﻧ ( ياﺮـﺑ ﺐـﺳﺎﻨﻣ ﻛاو ﻪﻨﻴﻣز رد ﺮﺘﺸﻴﺑ تﺎﻌﻟﺎﻄﻣ ﺴ ﻦ دﻮﺑ يزﺎﺳ . ﻲﺳرﺮﺑ شور : ﺖـﺳا ﻪﺘﻓﺮﮔ مﺎﺠﻧا ﻲﺑﺮﺠﺗ ترﻮﺻ ﻪﺑ ﻲﺳرﺮﺑ ﻦﻳا . رد روژﺎـﻣ ﺎﻴﻧﺎﻤﺸـﻴﻟ ،تﻮﮕﻴﺘـﺳﺎﻣوﺮﭘ ﻪـﻠﺣﺮﻣ رد ﻂﻴﺤﻣ N.N.N ﻂﻴﺤﻣ رد ﺲﭙﺳ و RPMI1640 ﻪﺑ ﺪـﺷ هداد ﺖﺸـﻛ هﻮﺒﻧا ترﻮﺻ . ﻪـﻣادا رد DNA ﺎـﺑ ﻞـﮕﻧا ﻲﻣﻮـﻧژ ﺪﺷ جاﺮﺨﺘﺳا نﺎﺷﻮﺟ شور . ﺲﭙﺳ PCR نژ ﻲﺻﺎﺼـﺘﺧا يﺎﻫﺮﻤﻳاﺮﭘ ﺎﺑ p4 ﺪـﺷ مﺎـﺠﻧا . لﻮﺼـﺤﻣ PCR لژ يور زا ﻢﻳﺰﻧآ ﺮﻴﺛﺄﺗ ﺖﺤﺗ و ﺪﺷ يﺮﮕﻟﺎﺑﺮﻏ ﺐﻴﻛﺮﺗ ﻮﻧ ﺪﻴﻤﺳﻼﭘ رد و ﺪﺷ يزﺎﺳ ﺺﻟﺎﺧ زورﺎﮔآ ﺖﻓﺮﮔ راﺮﻗ هﺪﻨﻫد شﺮﺑ يﺎﻫ . ﻪﺘﻓﺎﻳ ﺎﻫ : ﺶﻨﻛاو PCR ﻪـﻌﻄﻗ نﻮـﻠﻛ ﺲﭙـﺳ و p4 رد T-Vector ﺪـﺷ مﺎـﺠﻧا ﻲﺘـﺳرد ﻪـﺑ . ﻪـﻌﻄﻗ يوﺎـﺣ ﺪﻴﻤـﺳﻼﭘ ﻢﻳﺰﻧآ ﺎﺑ نآ رد هﺪﺷ نﻮﻠﻛ نژو جاﺮﺨﺘﺳا نﺎﻴﺑ ﺪﻤﻴﺳﻼﭘ و ﺪﺷ اﺪﺟ ،هﺪﻨﻫد شﺮﺑ يﺎﻫ PQE-30 نﻮـﻠﻛ بﺎـﺳ ﺖﻴﻘﻓﻮﻣ ﺎﺑ ﻢﻳﺰﻧآ ﻂﺳﻮﺗ ﺲﭙﺳ و ﺪﺷ مﺎﺠﻧا و هﺪﻨﻫد شﺮﺑ يﺎﻫ PCR ﺪﺷ ﺪﻴﻳﺄﺗ ،نﺎﻴﺑ ﺪﻴﻤﺳﻼﭘ يور . ﻪﺠﻴﺘﻧ يﺮﻴﮔ : ا نژ ،نﺎﻴﺑ هدﺎﻣ ﺐﻴﻛﺮﺗ ﻦﻳ p4 ﺖﺳا هﺎﮕﺸﻳﺎﻣزآ رد . ﺐﻴﻛﺮﺗ ﻮﻧ نژ ﻲﺘﻧآ ﺪﻴﻟﻮﺗ P4 ﻲﻣ ﺑ ،ﺪﻧاﻮﺗ ﻪ رد صﻮﺼﺧ ﺴﻛاو ﺔﻴﻬﺗ ﺔﻨﻴﻣز ﻦ ، ﻪﺑ رﺎﻛ ﻪﻨﻴﻣز رد نآ ﺮﺑ هوﻼﻋ و ﺪﻳآ ﺖﺴﺗ و ﻲﻳوراد فﺪﻫ يﺎﻫ ﺖﺳا هدﺎﻔﺘﺳا ﻞﺑﺎﻗ ﺰﻴﻧ ﻲﺼﻴﺨﺸﺗ يﺎﻫ . هژاوﺪﻴﻠﻛ ﺎﻫ : 1 نژ ـ P4 ، 2 ـ ،ﮓﻨﻴﻧﻮﻠﻛ 3 ،روژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ ـ 4 ﻦﺴﻛاو ـ
ﻪﻣﺪﻘﻣ
هداﻮﻧﺎـﺧ زا يرﺎـﺒﺟا ﻲﻟﻮﻠـﺳ ﻞـﺧاد ﻞﮕﻧا ﻚﻳ ،ﺎﻴﻧﺎﻤﺸﻴﻟ ﺖﺳا هﺪﻴﻣوزﻮﻧﺎﭙﻳﺮﺗ . دﻮﺟو ﻪﻠﺣﺮﻣ ود نآ ﻲﮔﺪﻧز ﻞﻜﻴﺳ رد دراد : ﻪﺸـﭘ نﺪـﺑ نورد ﻪـﻛ تﻮﮕﻴﺘـﺳﺎﻣوﺮﭘ راﺪﻛژﺎﺗ ﻪﻠﺣﺮﻣ ﻪــﻠﺣﺮﻣ و ﺖــﺳا ﻲﻜﻳژﻮــﻟﻮﻴﺑ ﻞــﻗﺎﻧ ناﻮــﻨﻋ ﻪــﺑ ﻲﻛﺎــﺧ هﺮـﻬﻣ نﺎـﻧﺎﺑﺰﻴﻣ يﺎـﻫژﺎﻓوﺮﻛﺎﻣ نورد ﻪـﻛ تﻮﮕﻴﺘﺳﺎﻣآ راد ﺖــﺳا . ) 1 ( ﻪــﻧﻮﮔ زا ﻲﻌﻴــﺳو ﻒــﻴﻃ ،ﺎﻴﻧﺎﻤﺸــﻴﻟ ﻒــﻠﺘﺨﻣ يﺎــﻫ يرﺎﻤﻴﺑ مﺮﻓ ﺎﺗ هﺪﻧﻮﺷ دوﺪﺤﻣ دﻮﺧ ﻲﺘﺳﻮﭘ يرﺎﻤﻴﺑ زا ار ﺎﻫ ﻲﻣ ﺐﺒﺳ ﻲﻳﺎﺸﺣا يرﺎﻤﻴﺑ ﺎﻳ ﺪﻳﺪﺷ هﺮﺸﺘﻨﻣ ﺪﻧﻮﺷ . ﺖﻴﻘﻓﻮﻣ نﺎﻣرد مﺪﻋ ﻞﻴﻟد ﻪﺑ ﺎـﻫوراد ﻲﺒﻧﺎﺟ ضراﻮﻋ ،ﺰﻴﻣآ ـﻛاو ﻚـﻳ ﻪـﻴﻬﺗ ،ﻲﻳوراد ﺖﻣوﺎﻘﻣ و ﺴ ﻪـﺟﻮﺗ درﻮـﻣ ﺐـﺳﺎﻨﻣ ﻦ ا ﻪﺘﻓﺮﮔ راﺮﻗ ﺮﺘﺸﻴﺑ ﺖﺳ . ) 2 ( ﻞﮕﻧا تﻮﮕﻴﺘﺳﺎﻣآ مﺮﻓ ﻪﻛ ﻲﻳﺎﺠﻧآ زا ﻲــﻣ نﺎﺴــﻧا رد يرﺎــﻤﻴﺑ ﺐﺒــﺳ ،دﻮــﺷ ﻲــﺘﻧآ ﻦﻳاﺮﺑﺎــﻨﺑ نژ يﺎــﻫ ﻛاو ﻪﻴﻬﺗ ﺖﻬﺟ ﻪﻠﺣﺮﻣ ﻦﻳا ﻲﺻﺎﺼﺘﺧا ﺴ ﻪـﺟﻮﺗ درﻮﻣ ﺮﺘﺸﻴﺑ ﻦ ﺪﻨﺘﺴﻫ ) 5 -3 ( . ﺪـﻴﻟﻮﺗ ،ﻲـﻠﺒﻗ تﺎـﻴﺑﺮﺠﺗ 8 يدﺎـﺑ ﻲـﺘﻧآ لﺎـﻧﻮﻠﻛﻮﻨﻣ يﻮﻧﺎﻔﻴﭘ ﺎﻴﻧﺎﻤﺸﻴﻟ ﻞﮕﻧا تﻮﮕﻴﺘﺳﺎﻣآ ﻪﻠﺣﺮﻣ ياﺮﺑ ار ﻲﺻﺎﺼﺘﺧا د شراﺰﮔ ﺪﻧدا ) p1 ﺎﺗ p8 .( ﻲﺘﻧآ لﺎﻧﻮﻠﻛﻮﻨﻣ يدﺎﺑ يﺎﻫ p4, p2 و p8 او ﺰﻴﻧ ﺲﻴﺴﻨﻧ وزﺎﻣآ ﺎﻴﻧﺎﻤﺸﻴﻟ تﻮﮕﻴﺘﺳﺎﻣآ ﺔﻠﺣﺮﻣ ﺎﺑ ـﻛ ﺶﻨ ﺪﻧداد نﺎﺸﻧ ) 4 ( . ﻲﺘﻧآ لﺎﻧﻮﻠﻛﻮﻨﻣ يدﺎﺑ يﺎﻫ p4 و p7 ﺪـﻧﺎﺑ ياراد دوﺪـــﺣ نزو ﺎـــﺑ ﻲــﻨﻴﺌﺗوﺮﭘ 34 ﺪﻨﺘﺴـــﻫ نﻮـــﺘﻟاد ﻮـــﻠﻴﻛ . ) 4 و 5 ( ﻲــﺘﻧآ نژ ﻂــﺳﻮﺗ ﺰــﻴﻧ تﻮﮕﻴﺘــﺳﺎﻣآ ﺔــﻠﺣﺮﻣ ﻲﺻﺎﺼــﺘﺧا يﺎــﻫ ﻲﺘﻧآ لﺎﻧﻮﻠﻛﻮﻨﻣ ﻦﻴﻤﻫ يدﺎﺑ ﻲﻣ ﻲﻳﺎﺳﺎﻨﺷ ﺎﻫ ﺪﻧﻮﺷ . زا ماﺪـﻛ ﺮﻫ ﻲﺘﻧآ ﻦﻳا نژ ﻪـﻴﻬﺗ ﺪـﻳﺪﻧﺎﻛ ناﻮـﻨﻋ ﻪـﺑ هﻮﻘﻟﺎـﺑ ﻞﻴﺴـﻧﺎﺘﭘ ﻚـﻳ ﺎـﻫ ﻲﻣ رﺎﻤﺷ ﻪﺑ ﻞﮕﻧا لﺮﺘﻨﻛ و يﺮﻴﮕﺸﻴﭘ ﺖﻬﺟ رد ﻦﺴﻛاو ﺪـﻨﻳآ . ) 5 ( ﻲﺘﻧآ نژ يﺎﻫ p4 و p8 تﻮﮕﻴﺘـﺳﺎﻣآ ﺔـﻠﺣﺮﻣ ﻚـﻴﻧﺰﮔآ ﺖﺸﻛ زا ﻪﺻﻼﺧ ﻪﻟﺎﻘﻣ ﻦﻳا نﺎﻳﺎﭘ زا ﺖﺳا يا ﻪﻣﺎﻧ ﺮﺘﻛد ﻞﮕﻧا ياﺮﺘﻛد ﻪﺟرد ﺖﻓﺎﻳرد ﺖﻬﺟ لددﺎﺷ ﻮﻨﻴﻣ و ﻲـﻗﻼﺧا ﻊـﻣﻻ ﺮـﺘﻛد و يدﺰـﻣروا دﺰـﻣﺮﻫ ﺮـﺘﻛد ﻲﻳﺎـﻤﻨﻫار ﻪـﺑ ﻲـﺳﺎﻨﺷ ﻲﻤﻇﺎﻛ ماﺮﻬﺑ ﺮﺘﻛد هروﺎﺸﻣ . (I ﻞﮕﻧا رﺎﻳدﺎﺘﺳا ﻞﮕﻧا هوﺮﮔ ،ﻲﺳﺎﻨﺷ چرﺎﻗ و ﻲﺳﺎﻨﺷ ازدﺎﻤﺘﻋا ﺪﻴﻬﺷ نﺎﺑﺎﻴﺧ ،ﻲﻤﻃﺎﻓ ﺪﻴﻬﺷ نﺎﺑﺎﻴﺧ ،ﺶﺗرا ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲﻜﺷﺰﭘ هﺪﻜﺸﻧاد ،ﻲﺳﺎﻨﺷ هد ، هﺎﮕﺸﻧاد ناﺮﻳا ،ناﺮﻬﺗ ،ﺶﺗرا ﻲﻜﺷﺰﭘ مﻮﻠﻋ *) لوﺆﺴﻣ ﻒﻟﺆﻣ ( (II ﻞﮕﻧا دﺎﺘﺳا ﻞﮕﻧا هوﺮﮔ ،ﻲﻜﺷﺰﭘ ﻲﺳﺎﻨﺷ چرﺎﻗ و ﻲﺳﺎﻨﺷ ﻲﻧﺎﻣرد ﻲﺘﺷاﺪﻬﺑ تﺎﻣﺪﺧ و ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲﻜﺷﺰﭘ هﺪﻜﺸﻧاد ،ﻲﺳﺎﻨﺷ -ناﺮﻳا ،ناﺮﻬﺗ ،-ناﺮﻳا (III ﻞﮕﻧا رﺎﻴﺸﻧاد ﻞﮕﻧا هوﺮﮔ ،ﻲﺳﺎﻨﺷ چرﺎﻗ و ﻲﺳﺎﻨﺷ ﭘ هﺪﻜﺸﻧاد ،ﻲﺳﺎﻨﺷ ﻲﺘﺷاﺪﻬﺑ تﺎﻣﺪﺧ و ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲﻜﺷﺰ -ناﺮﻳا ،ناﺮﻬﺗ ،-ناﺮﻳا ﻲﻧﺎﻣرد (IV ﻞﮕﻧا دﺎﺘﺳا ﻞﮕﻧا هوﺮﮔ ،ﻲﺳﺎﻨﺷ چرﺎﻗ و ﻲﺳﺎﻨﺷ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲﻜﺷﺰﭘ هﺪﻜﺸﻧاد ،ﻲﻟﻮﻜﻟﻮﻣ و ﻲﻟﻮﻠﺳ يژﻮﻟﻮﻴﺑ تﺎﻘﻴﻘﺤﺗ ﺰﻛﺮﻣ ،ﻲﺳﺎﻨﺷ ﻲﺘـﺷاﺪﻬﺑ تﺎﻣﺪـﺧ و ﻲﻧﺎـﻣرد ناﺮﻳا ،ناﺮﻬﺗ ،ﻲﺘﺸﻬﺑ ﺪﻴﻬﺷ (V ﺲﻧﺎﺴﻴﻟ قﻮﻓ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ،ﻲﻟﻮﻜﻟﻮﻣ و ﻲﻟﻮﻠﺳ يژﻮﻟﻮﻴﺑ تﺎﻘﻴﻘﺤﺗ ﺰﻛﺮﻣ ،يژﻮﻟﻮﻴﺑوﺮﻜﻴﻣ ﻲﺘﺷاﺪﻬﺑ تﺎﻣﺪﺧ و ﻲﻧﺎﻣرد ناﺮﻳا ،ناﺮﻬﺗ ،ﻲﺘﺸﻬﺑ ﺪﻴﻬﺷ
ﻲﻣ ﺺﻟﺎﺧ يﻮﻧﺎﻔﻴﭘ ﺎﻴﻧﺎﻤﺸﻴﻟ ﻞـﺑﺎﻗ ﺶـﺨﺑ ﺖـﻇﺎﻔﺣ و ﺪﻧﻮﺷ ﻪـــﻈﺣﻼﻣ ﻲـــﻣ ءﺎـــﻘﻟا ار يا ﺪـــﻨﻛ ) 3 و 5 ( . زا ﻲـــﻜﻳ ﻦﻳاﺮﺑﺎـــﻨﺑ ﻲﺘﻧآ نژ ،تﻮﮕﻴﺘـﺳﺎﻣآ مﺮـﻓ ﻲﺤﻄـﺳ يﺎﻫ p4 ـﺳا رد ﻪـﻛ ﺖ ﻦﺴﻛاو ﺖﻓﺮﺸﻴﭘ ﻪﻨﻴﻣز ﻲﻣ يزﺎﺳ ﺪﺷﺎﺑ ﺮﺛﺆﻣ ﺪﻧاﻮﺗ ) 5 ـ 3 ( . نژ ﻲــﺘﻧآ ةﺪــﻨﻨﻛ ﺪــﻛ نژ p4 و نﻮــﻠﻛ يﻮﻧﺎــﻔﻴﭘ ﺎﻴﻧﺎﻤﺸــﻴﻟ رد نژ ﺖﻫﺎﺒﺷ و ﺪﻳدﺮﮔ ﺲﻧﺎﻜﺳ p4 ﻪﻧﻮﮔ ﺮﻳﺎﺳ ﺎﺑ ﺲﻨـﺟ يﺎـﻫ ﺪـــﺷ ﻲـــﺳرﺮﺑ ،ﺎﻴﻧﺎﻤﺸـــﻴﻟ . ﻲـــﺘﻧآ ﻦـــﻳا ﻪﻜﺒـــﺷ نورد نژ ﺖــﻴﻟﺎﻌﻓ ياراد و دراد راﺮــﻗ تﻮﮕﻴﺘــﺳﺎﻣآ ﻚﻴﻤــﺳﻼﭘوﺪﻧآ ا يزﺎﺌﻠﻛﻮﻧ تﺎـﺒﺛ رد نآ ﻲﻟﺎﻤﺘﺣا ﺶﻘﻧ ﻪﻛ ﺖﺳ RNA ) نﺎـﻴﺑ نژ ( ﻢﻴﻣﺮﺗ و DNA ﺖﺳا ﺚﺤﺑ درﻮﻣ . ) 3 ( نژ ﻪـﻛ ﺖـﺳا ﻦـﻳا ﺮـﺑ ﺪﺼـﻗ ﻲﺳرﺮﺑ ﻦﻳا رد p4 ﺎﻴﻧﺎﻤﺸـﻴﻟ دﻮﺷ نﻮﻠﻛ ،ﺐﻴﻛﺮﺗ ﻮﻧ ﻦﻴﺌﺗوﺮﭘ ناﻮﻨﻋ ﻪﺑ ناﺮﻳا ﺔﻳﻮﺳ روژﺎﻣ .
ﻲﺳرﺮﺑ شور
ﺖــﺳا ﻪــﺘﻓﺮﮔ مﺎــﺠﻧا ﻲــﺑﺮﺠﺗ ترﻮــﺻ ﻪــﺑ ﻲــﺳرﺮﺑ ﻦــﻳا . تﻮﮕﻴﺘﺳﺎﻣوﺮﭘ روژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ يﺎﻫ (MRHO/IR/75/ER) رد ﻂﻴـــﺤﻣ N.N.N ﻂﻴـــﺤﻣ رد ﺲﭙـــﺳ و ﺪﻧﺪـــﺷ هداد ﺪـــﺷر RPMI 1640 ﺪﻧﺪﻴﺳر هﻮﺒﻧا ﺖﺸﻛ ﻪﺑ ) 1 ( . تﻮﮕﻴﺘـﺳﺎﻣوﺮﭘ ﺎـﺑ ﺎـﻫ ﻊﻤﺟ ندﺮﻛ ژﻮﻔﻳﺮﺘﻧﺎﺳ صﻮﺼـﺨﻣ ﺮﻓﺎﺑ ﻂﺳﻮﺗ و ﺪﻧﺪﺷ يروآ ) ﺰﻛﻮــﻠﮔ 33 / 0 لﻮــﻣ (M) ﺲﻳﺮــﺗ ، mM 10 ، mM 5 MgCl2 ، نﻮﺘﻳﺮﺗ -x 100 2 % ( ،نﺪﻧﺎـﺷﻮﺟ شور ﺎـﺑ و ﺪﻧﺪﺷ ﺰﻴﻟ DNA ﻲﻣﻮﻧژ نآ ﺪﺷ جاﺮﺨﺘﺳا ﺎﻫ ) 6 ( . نژ ﺲﻧﺎﻜـﺳ سﺎـﺳاﺮﺑ p4 ﻚـﻳ ﺖﻤـﺳ رد ﻪﻛ يرﻮﻃ ﻪﺑ ،ﺪﺷ ﻲﺣاﺮﻃ ﺮﻤﻳاﺮﭘ ﺖﻔﺟ 5 ﺎـﻫﺮﻤﻳاﺮﭘ forward و reverse ﻢﻳﺰﻧآ شﺮﺑ هﺎﮕﻳﺎﺟ BamHI ﺪﺷﺎﺑ . ﺶﻨــــﻛاو PCR ﻞﻣﺎــــﺷ : 1 مﺮــــﮔوﺮﻜﻴﻣ ) μg ( DNA ، picomol 20 يﺎـــــﻫﺮﻤﻳاﺮﭘ زا ﻚـــــﻳ ﺮـــــﻫ زا forward ، 0.4mM dNTPs, 4mM MgC12 ﺮﻓﺎــﺑ PCR 1x ، 25 / 1 ﺪﺣاو
(Cina Gen, Iran) DNA Taq Polymerase
ﻢﺠﺣ ﻪﻛ ﺎﺑ نآ dH20 ﻪﺑ 30 ،دﻮـﺑ هﺪـﺷ هﺪﻧﺎﺳر ﺮﺘﻴﻟوﺮﻜﻴﻣ ) ﻪـﻣﺎﻧﺮﺑ هﺎﮕﺘﺳد PCR ترﻮﺻ ﻪﺑ ؛ ﺎﻣد 94 ﻲﺘﻧﺎـﺳ ﻪﺟرد داﺮـﮔ (◦C) تﺪﻣ ﻪﺑ 10 ،ﻪﻘﻴﻗد 35 ﻞﻣﺎـﺷ ﻚـﻳ ﺮـﻫ ﻪـﻛ ﻞﻜﻴـﺳ : ﻪـﻠﺣﺮﻣ denaturation رد يﺎﻣد ◦C 94 تﺪـﻣ ﻪﺑ 40 ﻪـﻠﺣﺮﻣ ،ﻪـﻴﻧﺎﺛ annealing يﺎــﻣد ◦C 5 / 62 تﺪــﻣ ﻪــﺑ 60 ﻪــﻠﺣﺮﻣ و ﻪــﻴﻧﺎﺛ extention يﺎﻣد ◦C 72 تﺪﻣ ﻪﺑ 60 نﺎﻳﺎﭘ رد و ،ﺖﺳا ﻪﻴﻧﺎﺛ يﺎﻣد ◦C 72 تﺪـﻣ ﻪﺑ 5 ﺖـﻓﺮﮔ مﺎـﺠﻧا ﻪـﻘﻴﻗد ( ) 7 ( . لﻮﺼـﺤﻣ PCR زورﺎﮔآ لژ رد 5 / 1 % مﻮﻳﺪـﻴﺗا ﺎـﺑو ﺪـﺷ زرﻮـﻓوﺮﺘﻜﻟا ﺪﺷ يﺰﻴﻣآ ﮓﻧر ﺪﻳﺎﻣوﺮﺑ . ﺪﻧﺎﺑ DNA رﻮـﻧ ﺮـﻳز ﺮـﻈﻧ درﻮﻣ ﺶﻔﻨـــﺑ ءاروﺎـــﻣ (Ultraviolet- UV) ﺪـــﻳدﺮﮔ هﺪﻫﺎﺸـــﻣ ، ) 8 ( . لﻮﺼــﺤﻣ PCR زورﺎــﮔآ لژ رد 1 %
Low Melting Point
رﻮﻧ ﺮﻳز و ﺪﺷ زرﻮﻓوﺮﺘﻜﻟا UV ﺪﻧﺎﺑ DNA لژ زا ﺮـﻈﻧ درﻮﻣ ﺺﻟﺎﺧ و اﺪﺟ ﺪـﺷ يزﺎﺳ ) 9 ( . DNA شور ﻲـﻃ هﺪـﺷ ﺺﻟﺎـﺧ T/A Cloning ﺪﻴﻤﺳﻼﭘ نورد pBluescript ﺪﺷ نﻮﻠﻛ . ﺪﻴﻤﺳﻼﭘ ،ﻪﺻﻼﺧ ترﻮﺻ ﻪﺑ pBluscript ﺰـﻧآ ﺎـﺑ ﻪﻛ ﻢﻳ EcoRV شﺮﺑ مﺮﻔﺴـﻧاﺮﺗ ﻞﻳﺪﻴﺗﻮﺋﻮﻠﻛﻮﻧ ﻢﻳﺰﻧآ ﺎﺑ ،ﺪﺷ هداد ﻪﺑ يﺎﻬﺘﻧا ' 3 نآ dTTp ﺪﺷ ﻪﻓﺎﺿا ) 10 ( . ﺶﻨــﻛاو لﻮﺼــﺤﻣ Ligation ) ﺪــﻧﻮﻴﭘ ( ياﺮﻳﺬــﭘ لﻮﻠــﺳ رد E.coli ـ شﻮﺳ XL1 blue ﺪﺷ مرﻮﻔﺴﻧاﺮﺗ ) 11 ( رﺎﮔآ ﺖﻴﻠﭘ رد و LB يوﺎـــﺣ ﻪـــﻛ μg/ml 50 ) ﻲـــﻠﻴﻣ رد مﺮـــﮔوﺮﻜﻴﻣ ﺮـــﺘﻴﻟ ( ﻲﭙﻣآ ﺪـﺷ ﺶـﺨﭘ ،دﻮـﺑ ﻦﻴﻠﻴﺳ . ﻛ ﻲـﻨﻠ ﻂـﺳﻮﺗ ﺎـﻫ IPTG, X-gal ﻲﻨﻠﻛ و ﺪﻧﺪﺷ يﺮﮕﻟﺎﺑﺮﻏ ،ﺐـﻴﻛﺮﺗ ﻮـﻧ ﺪﻴﻤﺳﻼﭘ ياراد ﺪﻴﻔﺳ يﺎﻫ ﺪﻧﺪﺷ بﺎﺨﺘﻧا ) 12 ( . جاﺮﺨﺘـﺳا ،ﻦﻴﻟﺎﻜﻟا شور ﺎﺑ ﺐﻴﻛﺮﺗﻮﻧ ﺪﻴﻤﺳﻼﭘ ﺪﺷ ) 13 ( ﻢﻳﺰﻧآ ﺎﺑ و BamHI ﺪﻳدﺮﮔ ﻢﻀﻫ . ﺪﻧﺎﺑ DNA هﺪـﺷ دازآ نﺎﻴﺑ ﺪﻴﻤﺳﻼﭘ نورد و ﺪﺷ يزﺎﺳ ﺺﻟﺎﺧ pQE-30 ﻢﻳﺰﻧآ ﺎﺑ ﻪﻛ BamHI ﺷ هداد شﺮﺑ ﺪﻳدﺮﮔ نﻮﻠﻛ بﺎﺳ ،دﻮﺑ هﺪ . ﺪﺷ مرﻮﻔﺴﻧاﺮﺗ ،ﺶﻨﻛاو لﻮﺼﺤﻣ . ﻲﻨﻠﻛ ﺪﻴﻤـﺳﻼﭘ يوﺎـﺣ يﺎﻫ ﻂﻴـــﺤﻣ رد ﺐـــﻴﻛﺮﺗﻮﻧ LB ﺲﭙـــﺳ و ﺪـــﺷ هداد هﻮـــﺒﻧا ﺖﺸـــﻛ ، ﺪﻴﻤﺳﻼﭘ ﻂـﺳﻮﺗ ﻞـﻴﻠﺤﺗ و ﻪﻳﺰﺠﺗ ﺎﺑ و ﺪﺷ جاﺮﺨﺘﺳا ﺐﻴﻛﺮﺗﻮﻧ يﺎﻫ ﻢﻳﺰﻧآ ﺪـﺷ ﺪـﻴﻳﺄﺗ هﺪـﻨﻫد شﺮﺑ يﺎﻫ . ﺶﻨـﻛاو PCR زا هدﺎﻔﺘـﺳا ﺎـﺑ ﺮﻤﻳاﺮﭘ forward Universal و reverse نژ ﻲﺻﺎﺼﺘﺧا p4 ﺖـﻬﺟ ﺪﻴﻳﺄﺗ orientation ) ﺖﻬﺟ ( ﺖﻓﺮﮔ مﺎﺠﻧا ،هﺪﺷ نﻮﻠﻛ نژ ﺐﺳﺎﻨﻣ .
ﻪﺘﻓﺎﻳ
ﺎﻫ
ﻂﻴﺤﻣ رد ﺎﻴﻧﺎﻤﺸﻴﻟ تﻮﮕﻴﺘﺳﺎﻣوﺮﭘ N.N.N رد و ﻪﺘﻓﺎﻳ ﺪﺷر ﻂﻴﺤﻣ RPMI 1640 1640 و ﺪـﺷ هداد ﺖﺸـﻛ هﻮـﺒﻧا ترﻮﺻ ﻪﺑ DNA شور ﺎﺑ و هﺪﺷ جاﺮﺨﺘﺳا نآ ﻲﻣﻮﻧژ PCR ﺖﻓﺎﻳ ﺮﻴﺜﻜﺗ . DNA ـﺷ رد هﺪـﺷ جاﺮﺨﺘﺳا هرﺎﻤـﺷ ﻞﻜ 1 هداد نﺎﺸـﻧ
ﺖﺳا هﺪﺷ . هرﺎﻤـﺷ ﻞﻜـﺷ رد 2 لﻮﺼـﺤﻣ PCR نژ p4 ﺎـﺑ نزو bp 965 ﺖﺳا هﺪﺷ هداد نﺎﺸﻧ . هرﺎﻤﺷ ﻞﻜﺷ 1 زرﻮﻓوﺮﺘﻜﻟا DNA زرﺎﮔآ لژ رد ﺎﻴﻧﺎﻤﺸﻴﻟ ﻲﻣﻮﻧژ 1 % ﻞﻜﺷ هرﺎﻤﺷ 2 لﻮﺼﺤﻣ زرﻮﻓوﺮﺘﻜﻟا PCR زرﺎﮔآ لژ رد 1 % نﻮﺘﺳ I : ﺮﻛرﺎﻣ 100bp DNA نﻮﺘﺳ II : لﻮﺼﺤﻣ PCR نژ p4 نزو ﺎﺑ bp 965 لﻮﺼﺤﻣ PCR ﻞـﻗﺎﻧ ﺪﻴﻤﺳﻼﭘ رد pBluescript شور ﺎـﺑ ﮓﻨﻴﻧﻮﻠﻛ T/A ﺪـﺷ نﻮﻠﻛ . ﻢﻳﺰـﻧآ ﻂـﺳﻮﺗ ،ﺐـﻴﻛﺮﺗﻮﻧ ﺪﻴﻤـﺳﻼﭘ هﺪﻨﻫد شﺮﺑ BamHI ﺪﻧﺎﺑ و ﺪﺷ هداد شﺮﺑ DNA نژ p4 اﺪﺟ نﺎﻴﺑ ﺪﻴﻤﺳﻼﭘ رد و ﺪﺷ pQE-30 ﻢﻳﺰـﻧآ ﺎـﺑ ًﻼﺒـﻗ ﻪﻛ BamHI ﺪــﺷ نﻮــﻠﻛ بﺎــﺳ ،دﻮــﺑ هﺪــﺷ هداد شﺮــﺑ . ﺶﻨــﻛاو Ligation ) ﺪﻧﻮﻴﭘ ( ﺖﻴﻠﭘ ردو ﺪﺷ مرﻮﻔﺴﻧاﺮﺗ ﻲﻳﺎﻳﺮﺘﻛﺎﺑ ياﺮﻳﺬﭘ لﻮﻠﺳ رد رﺎﮔآ LB يوﺎﺣ μg/mL 50 ﻲﭙﻣآ ﺪﺷ ﺶﺨﭘ ﻦﻴﻠﻴﺳ . ﺪﻴﻤـﺳﻼﭘ شﺮﺑ ﻢﻳﺰﻧآ ﺎﺑ ﻞﻴﻠﺤﺗ و ﻪﻳﺮﺠﺗ ﻂﺳﻮﺗ ،ﺐﻴﻛﺮﺗﻮﻧ هﺪـﻨﻫد pmacl نژ رد ﻲﺻﺎﺼـﺘﺧا شﺮـﺑ هﺎﮕﻳﺎﺟ ﻪﻛ p4 ﺪﻴﻤـﺳﻼﭘ و دراد ار ﻲﻤﻧ شﺮﺑ ار ﺐﻴﻛﺮﺗﻮﻧ ﺮﻴﻏ ﺪﺷ ﺪﻴﻳﺄﺗ ،ﺪﻫد . هرﺎﻤــﺷ ﻞﻜــﺷ 3 ﻢﻳﺰــﻧآ ﺎــﺑ ﻪــﻛ ار ﺐــﻴﻛﺮﺗﻮﻧ ﺪﻴﻤــﺳﻼﭘ pmacl ﻲﻣ نﺎﺸﻧ ،ﺖﺳا هﺪﺷ ﻲﻄﺧ و هﺪﺷ شﺮﺑ ﺪﻫد . ﻞﻜﺷ هرﺎﻤﺷ 3 -ﺐﻴﻛﺮﺗﻮﻧ ﺪﻴﻤﺳﻼﭘ ﺪﻴﺋﺎﺗ نﻮﺘﺳ I : ﻢﻟﺎﺳ ﺪﻴﻤﺳﻼﭘ pQE-30 ﻢﻳﺰﻧآ ﺮﻴﺛﺄﺗ زا ﻞﺒﻗ نﻮﺘﺳ II : ﻢﻟﺎﺳ ﺪﻴﻤﺳﻼﭘ pQE-30 ﻢﻳﺰﻧآ ﺮﻴﺛﺄﺗ زا ﺪﻌﺑ نﻮﺘﺳ III : راد ﻪﻌﻄﻗ ﺪﻴﻤﺳﻼﭘ pQE-30-4 ﺛﺄﺗ زا ﺪﻌﺑ ﻢﻳﺰﻧآ ﺮﻴ نﻮﺘﺳ IV : راد ﻪﻌﻄﻗ ﺪﻴﻤﺳﻼﭘ pQE-30-4 ﻢﻳﺰﻧآ ﺮﻴﺛﺄﺗ زا ﻞﺒﻗ هرﺎﻤﺷ ﻞﻜﺷ 4 لﻮﺼـﺤﻣ PCR ﺖـﻬﺟ ﻦﻴـﻴﻌﺗ ياﺮـﺑ (orientation) نژ ﺐﺳﺎﻨﻣ p4 ﺪﻴﻤﺳﻼﭘ رد هﺪﺷ نﻮﻠﻛ نﻮﺘــﺳ I : لﻮﺼــﺤﻣ PCR نژ P4 ) bp 1050 ( ﺮــﻤﻳاﺮﭘ زا هدﺎﻔﺘــﺳا ﺎــﺑ universal forward و specific p4 reverse نﻮﺘﺳ II : ﭘ ﺪﻴﻤﺳﻼ pQE-30-4 نﻮﺘﺳ III : ﺮﻛرﺎﻣ (bp) 100 DNA
مﺎﺠﻧا PCR ﺮـﻤﻳاﺮﭘ زا هدﺎﻔﺘﺳا ﺎﺑ forward Universal و reverse نژ ﻲﺻﺎﺼــــﺘﺧا p4 ﺖــــﻬﺟ ﻦﻴــــﻴﻌﺗ ياﺮــــﺑ (orientation) نﺎـﻴﺑ ﺪﻴﻤـﺳﻼﭘ رد هﺪـﺷ نﻮـﻠﻛ نژ ﺐﺳﺎﻨﻣ pQE-30 هرﺎﻤـﺷ ﻞﻜﺷ رد ﻪﻛ ﺪﺷ ﻪﺘﻓﺮﮔ رﺎﻛ ﻪﺑ 4 ﺶﻳﺎـﻤﻧ ﺖﺳا ﻪﺘﻓﺎﻳ .
ﺚﺤﺑ
ﻧﺎﻤﺸﻴﻟ ﻪـﻛ ﺖـﺳا يرﺎـﺒﺟا ﻲﻟﻮﻠـﺳ ﻞـﺧاد ﻞـﮕﻧا ﻚـﻳ ،ﺎﻴ ﺗ تﻮﮕﻴﺘﺳﺎﻣوﺮﭘ ﻪﻠﺣﺮﻣ ﺬﮔﺮﻴﺛﺄ ﻲﻛﺎـﺧ ﻪﺸـﭘ نﺪـﺑ رد ،نآ را نﺎــﺑﺰﻴﻣ نﺪــﺑ رد تﻮﮕﻴﺘــﺳﺎﻣآ ﻪــﻠﺣﺮﻣ و ﻞــﻗﺎﻧ ) هﺮــﻬﻣ راد ( دراد دﻮﺟو ) 1 ( . رد ﺖـﻴﻘﻓﻮﻣ مﺪـﻋ ﻞـﻴﻟد ﻪـﺑ ﺐـﺳﺎﻨﻣ ﻦﺴـﻛاو ﻚـﻳ ﻪﻴﻬﺗ و ﺎــﻫوراد ﻲﺒﻧﺎــﺟ ضراﻮــﻋ ،ﻲﺑﺎــﺨﺘﻧا و ﻲــﺳﺎﺳا نﺎــﻣرد ـــﺟﻮﺗ درﻮـــﻣ ،ﻲـــﺋوراد ﺖـــﻣوﺎﻘﻣ راﺮـــﻗ يﺮﺘﺸـــﻴﺑ ﻪ ﺖﺳا ﻪﺘﻓﺮﮔ . ) 2 و 3 ( نژ لﻮﺼﺤﻣ p4 ﻪـﻠﺣﺮﻣ ﻚﻴﻤـﺳﻼﭘوﺪﻧآ ﻪﻜﺒـﺷ رد ﻪـﻛ ﻦﺴـﻛاو ﺖﻓﺮﺸـﻴﭘ ﺮـﻣا رد ،دراد راﺮـﻗ تﻮﮕﻴﺘﺳﺎﻣآ يزﺎـﺳ ﺖــﺳا ﺐــﺳﺎﻨﻣ ) 3 ( . ﻪــﻛ ﺖــﺳا هداد نﺎﺸــﻧ تﺎــﻌﻟﺎﻄﻣ ﻲــﺧﺮﺑ ﻲــﺘﻧآ نژ p4 و دراد تﻮﮕﻴﺘــﺳﺎﻣآ ﻪــﻠﺣﺮﻣ ﻪــﺑ صﺎﺼــﺘﺧا ژﻮﻟﻮﻣﺪﻴﭘا ،يﺪﻨﺑ هدر ﻪﻨﻴﻣز رد نآ هﺪﺷ ﺺﻴﻠﺨﺗ ﻦﻴﺌﺗوﺮﭘ ي يژﻮﻟﻮﻧﻮﻤﻳا و ﻲﻣ ﻣ ﺪﻨﻧاﻮﺗ ﺆ ﺪﺷﺎﺑ ﺮﺛ ) 5 -2 ( . تﺎﻌﻟﺎﻄﻣ رد Sujata ﻪـﻛ ﺖـﺳا هﺪـﺷ ﺮﻛذ ﺶﻧارﺎﻜﻤﻫ و ﻲﺘﻧآ ﺺﻟﺎﺧ نژ p4 شﻮﻣ رد BALB/c ﺖـﻧﻮﻔﻋ ﻞـﺑﺎﻘﻣ رد ﻲﺸﺨﺑ ﺖﻇﺎﻔﺣ ،ﺲﻴﺴﻨﻧوزﺎﻣآ و يﻮﻧﺎﻔﻴﭘ ﺎﻴﻧﺎﻤﺸﻴﻟ زا ﻲﺷﺎﻧ عﻮـﻧ زا ﻲﻨﻤﻳا ﺦﺳﺎﭘ ءﺎﻘﻟا ﻦﻴﻨﭽﻤﻫ و ﻞﻣﺎﻛ ﺎﺗ ﻲﺒﺴﻧ Th1 ار ﺚـﻋﺎﺑ ﻲﻳﺎـﻜﻳﺮﻣآ يﺪﻠﺟ ﺲﻳزﻮﻴﻧﺎﻤﺸﻴﻟ ﻪﺑ ﻼﺘﺒﻣ نارﺎﻤﻴﺑ رد دﻮﺑ هﺪﺷ ) 3 ( . ﻞﻴﺴـﻧﺎﺘﭘ ﻲﺳرﺮﺑ ﺖﻬﺟ DNA ﻦﺴـﻛاو ،ﺎـﻫ Cambell و زﺎــــﺌﻠﻛﻮﻧ ةﺪــــﻨﻨﻛﺪﻛ نژ ﺶﻧارﺎــــﻜﻤﻫ p4 ﺎﻴﻧﺎﻤﺸــــﻴﻟ رد ﺖـﻧاﻮﺟدا ﺎﺑ هاﺮﻤﻫ ار ﺲﻴﺴﻨﻧوزﺎﻣآ ﻒـﻠﺘﺨﻣ يﺎـﻫ IL-12 و HSP70 سﺎﺴﺣ شﻮﻣو هدﺮﻛ ﻲﺑﺎﻳزرا BALB/c ﻂﺳﻮﺗ ار p4 ﻳﺎﻬﻨﺗ ﻪﺑ ،ﻲ p4 ﺎﺑ هاﺮﻤﻫ IL-12 و p4 ﺎـﺑ هاﺮـﻤﻫ HSP70 ﺎﻴﻧﺎﻤﺸــﻴﻟ ﺎــﺑ ﺖــﻧﻮﻔﻋ ضﺮــﻌﻣ رد ﺲﭙــﺳ و هدﺮــﻛ ﻦــﻤﻳا داد راﺮﻗ روژﺎﻣ و ﺲﻴﺴﻨﻧوزﺎﻣآ . نآ ﺎﻫ نژ ﻪـﻛ ﺪﻧداد نﺎﺸﻧ p4 ﺎــﺑ هاﺮــﻤﻫ IL-12 ﺲﻴﺴــﻨﻧوزﺎﻣآ ﺎﻴﻧﺎﻤﺸــﻴﻟ ﻞــﺑﺎﻘﻣ رد ﻲﻟو ﻪﺘﺷاد ﻲﺸﺨﺑ ﺖﻇﺎﻔﺣ p4 ﺎﺑ هاﺮﻤﻫ HSP70 ﻞـﺑﺎﻘﻣ رد ﻲﺸﺨﺑ ﺖﻇﺎﻔﺣ روژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ ﺖـﻳﺎﻬﻧ رد و ﺖـﺳا ﻪﺘـﺷاد p4 ﺎـﺑ هاﺮﻤﻫ HSP70 ﻪـﻴﻬﺗ ﺖـﻬﺟ ﺐـﺳﺎﻨﻣ ﺪـﻳﺪﻧﺎﻛ ﻚـﻳ ار ﻪـﻧﻮﮔ ياﺮـﺑ ﻦﺴﻛاو ،ﺪـﻳﺪﺟ و ﻢﻳﺪـﻗ يﺎـﻴﻧد ﺎﻴﻧﺎﻤﺸـﻴﻟ يﺎـﻫ دﺮﻛ ﻲﻓﺮﻌﻣ ) 2 ( . نژ ،ﻲﺳرﺮﺑ ﻦﻳا رد p4 ناﺮﻳا ﺔﻳﻮﺳ روژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ ﺪﺷ نﻮﻠﻛ . ﻲـﻧاﺮﻳا ﺔﻳﻮـﺳ زا هدﺎﻔﺘـﺳا ﻪـﻛ فﺪـﻫ ﻦـﻳا ﺎﺑ ژﺎﻣ ﺎﻴﻧﺎﻤﺸﻴﻟ ﻪﻴﻠﻋ ﺮﺑ ﻦﺴﻛاو ﺔﻴﻬﺗ ﺖﻬﺟ ﻪـﻌﻣﺎﺟ ياﺮﺑ رو يدﺮﺑرﺎﻛ و ﺮﺗﺪﻴﻔﻣ ،ﻲﻧاﺮﻳا ﺖﺳا ﺮﺗ . ﺔـﺠﻴﺘﻧ ﻪـﻜﻨﻳا ﻦﻤـﺿ جﺮﻓ ﻲﺳرﺮﺑ ﺎﺑ ﻲﺳرﺮﺑ ﻦﻳا شراﺰـﮔ ﻪـﻛ نارﺎـﻜﻤﻫ و ﺎـﻴﻧ هداد ﺪﻧا » سﻼـﻛ زﺎﺌﻠﻛﻮﻧ ﻦﻴﺌﺗوﺮﭘ 1 تﻮﮕﻴﺘـﺳﺎﻣآ ﺔـﻠﺣﺮﻣ روژﺎــﻣ ﺎﻴﻧﺎﻤﺸــﻴﻟ (Lma CIN) يدﺎــﻳز رﺎﻴﺴــﺑ ﺖﻫﺎﺒــﺷ ) 87 (% ﻦﻴﺌﺗوﺮــﭘ ﻪــﺑ p4 دراد يﻮﻧﺎــﻔﻴﭘ ﺎﻴﻧﺎﻤﺸــﻴﻟ « ) 14 و 15 ( ﻞﺑﺎﻗ دﻮﺑ ﺪﻫاﻮﺧ ﻪﺴﻳﺎﻘﻣ . ﻪــﻛ ﻲﻳﺎــﺠﻧآ زا ﻲــﺳرﺮﺑ ﻦــﻳا رد DNA ﺪــﻗﺎﻓ ﺎﻴﻧﺎﻤﺸــﻴﻟ ﺖــﺳا نوﺮــﺘﻨﻳا ) 16 ( نژ ﻪــﺑ ﻲــﺳﺮﺘﺳد ﺖــﻬﺟ ، p4 زا DNA ﺮـﻈﻧ ﻪـﺑ و ﺪـﺷ هدﺎﻔﺘـﺳا روژﺎـﻣ ﺎﻴﻧﺎﻤﺸـﻴﻟ تﻮﮕﻴﺘﺳﺎﻣوﺮﭘ ﻲﻣ رد ﻢـﺧز دﺎـﺠﻳا شور ﻪـﺑ ﺖﺒﺴـﻧ شور ﻦـﻳا ﻪـﻛ ﺪـﺳر ﻂﻴـﺤﻣ زا هدﺎﻔﺘـﺳا ﺎـﻳ و ﻲﻫﺎﮕﺸـﻳﺎﻣزآ ناﻮﻴﺣ ﺖﺸـﻛ يﺎـﻫ ﺎﺴـﺣ و ﻲﺻﺎﺼﺘﺧا ﻪـﺑ ﻲـﺳﺮﺘﺳد ﺖـﻬﺟ تﻮﮕﻴﺘـﺳﺎﻣآ س نژ ﻲﻣ نﺎﻴﺑ تﻮﮕﻴﺘﺳﺎﻣآ ﺔﻠﺣﺮﻣ رد ﻪﻛ ﻲﻳﺎﻫ ﻊﻳﺮﺳ ،ﺪﻧﻮﺷ ﺮﺗ هدﺎﺳ و ﺪﺷﺎﺑ ﺮﺗ ) 15 ( . ﻲﻟﻮﻜﻟﻮﻣ و ﻲﻟﻮﻠﺳ تﺎﻌﻟﺎﻄﻣ مﺎﺠﻧا رد ﺖﻳدوﺪﺤﻣ دﻮﺟو ﺪـﻳﺎﺑ اﺪـﺘﺑا ﺶﻫوﮋﭘ ﻞﺣاﺮﻣ ﻪﻛ ﻲﻄﻳاﺮﺷ رد هﮋﻳو ﻪﺑ ،ﻢﻴـﻈﻨﺗ زا ﻲــﻜﻳ ،ﺪﻧﻮــﺷ مﺎــﺠﻧا ﺲﭙــﺳ و ﺐــﺗﺮﻣ و ﻲﻫﺪﻧﺎﻣزﺎــﺳ ﻪـﻛ ﺖﺳا يﺪﺟ تﻼﻜﺸﻣ ﻦـﻳا ﺮـﻴﮔ رد ﺰـﻴﻧ ﺮـﺿﺎﺣ ﻖـﻴﻘﺤﺗ دﻮﺑ ﻞﻜﺸﻣ .
ﻪﺠﻴﺘﻧ
يﺮﻴﮔ
ﻪﺠﻴﺘﻧ و نﻮـﻠﻛ ،ﺶﻫوﮋـﭘ ﻦـﻳا مﺎـﺠﻧا زا ﻞـﺻﺎﺣ ﻲﻠﻛ
هدﺎﻣآ نژ يزﺎﺳ p4 ﺖﺳا نﺎﻴﺑ ﺪﻴﻤﺳﻼﭘ رد . ﺐـﻴﻛﺮﺗ ﻦـﻳا ﺐـﻴﻛﺮﺗ ﻮﻧ ﻦﻴﺌﺗوﺮﭘ ﻦﺘﺧﺎﺳ و هﺎﮕﺸﻳﺎﻣزآ رد نﺎﻴﺑ هدﺎﻣآ ﺖﺳا . ﺐـﻴﻛﺮﺗﻮﻧ ﻦﻴﺌﺗوﺮـﭘ ﺪـﻴﻟﻮﺗ ﻪـﻛ ﺖـﺳا ﺮﻛذ ﻞﺑﺎﻗ p4 ﻲﻣ رد صﻮﺼﺧ ﻪﺑ ﺪﻧاﻮﺗ ﻦﺴﻛاو ﺔﻴﻬﺗ ﺔﻨﻴﻣز ﻪﺑ ﺪـﻳآ رﺎﻛ و ﻪﻨﻴﻣز رد ،نآ ﺮﺑ هوﻼﻋ ﺖﺴﺗ و ﻲﻳوراد فﺪﻫ يﺎﻫ يﺎﻫ ﺖﺳا هدﺎﻔﺘﺳا ﻞﺑﺎﻗ ﺰﻴﻧ ﻲﺼﻴﺨﺸﺗ .
ﺮﻜﺸﺗ و ﺮﻳﺪﻘﺗ
و ﻲﻜـﺷﺰﭘ مﻮـﻠﻋ هﺎﮕﺸـﻧاد ﻲﻟﺎـﻣ ﺖـﻳﺎﻤﺣ ﺎـﺑ ﻲﺳرﺮﺑ ﻦﻳا تﺎــﻘﻴﻘﺤﺗ ﺰــﻛﺮﻣ هﺎﮕﺸــﻳﺎﻣزآ رد و ناﺮــﻳا ﻲﻧﺎــﻣرد تﺎﻣﺪــﺧ ﻲﻜﺷﺰﭘ مﻮﻠﻋ هﺎﮕﺸﻧاد ﻲﻟﻮﻜﻟﻮﻣ و ﻲﻟﻮﻠﺳ يژﻮﻟﻮﻴﺑ تﺎﻣﺪﺧ و نﺎﮔﺪﻨﺴـﻳﻮﻧ ﻪﻠﻴـﺳو ﻦﻳﺪﺑ و ﺪﺷ مﺎﺠﻧا ،ﻲﺘﺸﻬﺑ ﺪﻴﻬﺷ ﻲﻧﺎﻣرد زا ﻪﻟﺎﻘﻣ ﻟوﺆﺴﻣ ﺪﻧراد ار يراﺰﮕﺳﺎﭙﺳ لﺎﻤﻛ مﺮﺘﺤﻣ ﻦﻴ .
ﻊﺑﺎﻨﻣ ﺖﺳﺮﻬﻓ
1. Markell EK, John DT, Krotoski WA. Medical Parasitology. 8th ed. Philadelphia: Saunders WB; 1999.
P.184-59
2. Campbell K, Diao H, Jiaxiang J, Soong L. DNA Immunization with the Gene Encoding p4 Nuclease of Leishmaina amazonensis protects Mice against Cutaneous leishmaniasis. Infect Imuun 2003; 71(11):6270-8
3. Sujata K, Soong L, Colmenares M, Gold smith K, Mc Mahon-pratt K. The Immunologically protective p-4 Antigen of leishmania Amastigotes. J Biol Chem 2000; 275:37789-97
4. Pan AA, Mc Mahon-pratt D. Monoclonal antibodies specific for the amastigote stage of leishmania pifanoi. J Immun 1998; 140(7): 2406-14.
5. Hodgkinson VH, Soong L, Duboise SM, Mc Mahon-pratt D. Leishmania amazonensis: Cultivation and Characterization of Axenic Amastigote-like Organisms. Exp Parasit 1996; 83:94-105.
6. Brown TA. An Introduction to gene cloning. 3rded.
UK: International Thomson Publishing Services; 1995. P.32-41
7. Pherson Mc, Moller MJ PCR. The Basics from Background to Bench. Understanding PCR. 1st ed.
Oxford, UK: Bios Scientific publishers; 2000. P.9-21. 8. Boffey SA. Agarose gel electroiphoresis of DNA. Walker JM. Nucleic acids (Methods in Molecular Biology) Vol.2. 1st ed . USA: Humana Press; 1984. P.43-50.
9. Gaastra W, Jorgensen PL. The extraction and isolation of DNA from gels. Walker JM. Nucleic acids (Methods in Molecular Biology) (Vol.2). 1st ed. USA:
Humana Press; 1984. P.67-76.
10. Gaastra W. Hansen K. Ligation of DNA with T4 DNA ligase. Walker JM. Nucleic acids (Methods in Molecular Biology) (Vo1.2). 1st ed. USA: Humana
Press; 1984. P.225-30.
11. Hunahan D. Studies on transformation on E. coli with plasmids. J Mol Biol 1983;166(4):557-80
12. Bothwell AL, Yancopulos GD, Alt FW. Methods for cloning and analysis of eukaryotic genes. 1st ed.
Boston: Jones and Bartlett Publishers; 1990. P. 247-60. 13. Feliciecello I, Chinali G. A modified alkaline lysis method for the preparation of highly purified plasmid DNA form E. coli. Anal Biochem 1993: 212(2):394-401
14. Farajnia S, Mahboudi F, Ajdari S, Reiner NE, Kariminia A, Alimohammadian MH. Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to leishmania major amastigote class I nuclease with a predominant Thl-like response. Clin Exp Immunol 2005; 139:498-505.
15. Farajnia S, Alimohammadian MH, Reiner NE, Karimi M, Ajdari S, Mahboudi F. Molecular characterization of a novel amastigote stage specific Class 1 nuclease from Leishmania major. Int J Parasitol 2004; 34(8):899-908.
16. Fooladvand MA. Cloning and expression Leishmania major p36/Lack gene and evaluation the immunity of Lack Recombinant Protein. Thesis. Esfahan: Esfahan University of Medical Sciences and Health Services; 2006. P.7.
Evaluating the Cloning of Leishmania Major p4 Gene in Production
of Vaccine
*M. Shaddel, PhDI H. Ormazdi, PhDII L. Akhlaghi, PhDIII
B. Kazemi, PhDIV M. Bandepour, MScV
Abstract
Background and Aim: Leishmania major p4 gene is normally expressed during amastigote form of
the parasite and can be a good candidate for producing an effective vaccine. The aim of this study is to clone p4 gene in a suitable vector for further vaccine production studies .
Materials and Methods: This is an experimental study. Leishmania promastigote was grown in
N.N.N. medium and later in the form of mass culture in RPMI 1640 cell culture medium. Total leishmania genomic DNA was extracted by centrifugation of promastigote and lysed by lysis buffer followed by boiling method. PCR was carried out using p4 gene specific primers. PCR product was detected by agaros gel electrophoresis and cloned into Bluescript plasmid via T/A cloning method. After transformation, the recombinant plasmid was screened and digested by restriction enzyme
Results: PCR reaction and cloning p4 gene in T-vector was done successfully. Recombinant plasmid
was extracted and cloned gene was released by restriction enzyme and subcloned into pPQE-30 expression vector and confirmed by restriction analysis and PCR.
Conclusion: This newly expressed combination is p4 gene invitro. Production of p4 recombinant
antigen is very much useful especially in the field of vaccine production, drug target and immunological studies.
Key words: 1) p4 gene 2) Cloning 3) Leishmania major 4) Vaccine
This article is a Summary of the thesis by M. Shaddel for the degree of Ph.D. in Parasitology under supervision of H. Ormazdi, Ph.D. and L. Akhlaghi, Ph.D. and consultation with B. Kazemi, Ph.D.
I) Assistant Professor of Parasitology, Mycology and Parasitology Department, Faculty of Medicine, Shaheed Fatemi st., Shaheed Etemad Zadeh St., Army University of Medical sciences, Tehran, Iran (*Corresponding Author)
II) Professor of Medical Parasitology, Mycology and Parasitology Department, Faculty of Medicine, Iran University of Medical Sciences and Health Services Tehran, Iran
III) Associate Professor of Parasitology, Mycology and Parasitology Department, Faculty of Medicine, Iran University of Medical Sciences and Health Services Tehran, Iran
IV) Professor of Parasitology, Faculty of Medicine, Research Institute for Cellular and Molecular Biology, Shaheed Beheshti University of Medical Sciences and Health Services, Tehran, Iran
V) MSc in Microbiology, Research Institute for Cellular and Molecular Biology, Shaheed Beheshti University of Medical Sciences and Health Services, Tehran, Iran
