fa ‫ﻃﺮاﺣﯽ و ﺳﺎﺧﺖ ‪ BCG‬ﻧﻮﺗﺮﮐﯿﺐ ﺗﺮﺷﺢ ﮐﻨﻨﺪه ﭘﻠﯽﺗﻮپﻫﺎی وﯾﺮوس‬ ‫ﻫﭙﺎﺗﯿﺖ ‪ c‬ﺑﻪﻋﻨﻮان ﮐﺎﻧﺪﯾﺪای واﮐﺴﻦ‬ ویروس شناسی پزشکی Medical Virology مقاله پژوهشی Original Research Article <div align="right" style="direction: rtl"> ‫<b>زﻣﯿﻨﻪ واﻫﺪاف: </b>ﺑﯿﻤﺎری ﮐﺒﺪی ﻣﺮﺗﺒﻂ ﺑﺎ ﻋﻔﻮﻧﺖ ﻣﺰﻣﻦ وﯾﺮوس ﻫﭙﺎﺗﯿﺖ ‪  ،(HCV) c‬از ﻣﺸﮑﻼت ﻋﻤﺪه ﺑﻬﺪاﺷـﺖ ﺟﻬـﺎﻧﯽ‬ ‫اﺳﺖ. وﻟﯽ ﻫﻨﻮز واﮐﺴﻦ ﺗﺎﯾﯿﺪ ﺷﺪه ﻋﻠﯿﻪ ‪  HCV‬وﺟﻮد ﻧﺪارد. ﻣﺸﺨﺺ ﺷﺪه اﺳﺖ ﮐـﻪ ﭘﺎﺳـﺦﻫـﺎی اﯾﻤﻨـﯽ ﺳـﻠﻮﻟﯽ، ﺑـﻪ‬ ‫ﺧﺼﻮص ﭘﺎﺳﺦﻫﺎی ‪ CTL‬ ﻧﻘﺶ وﯾﮋهای در درﻣﺎن ﻋﻔﻮﻧﺖ ﺑﺎ اﯾﻦ وﯾﺮوس دارﻧﺪ. ﻧﻈﺮ ﺑﻪ اﯾﻨﮑﻪ واﮐﺴﻦ ‪ BCG‬ ﺗﻮاﻧﺎﯾﯽ‬ ‫ﺗﺤﺮﯾﮏ ﭘﺎﺳﺦ اﯾﻤﻨﯽ ﺳﻠﻮﻟﯽ را ﺑﺨﻮﺑﯽ دارد، ﻟﺬا در ﺗﺤﻘﯿﻖ ﺣﺎﺿﺮ ﺗﻼش ﺑﺮ اﯾﻦ ﺑﻮد ﮐﻪ ﺑﺎ ﺳﺎﺧﺖ ﮐﺎﻧﺪﯾـﺪای واﮐﺴﻦ‬ ‫‪ BCG‬ ﻧﻮﺗﺮﮐﯿﺐ (‪ (recombinant BCG=rBCG‬ﺗﺮﺷﺢ ﮐﻨﻨﺪه اﭘﯽ ﺗﻮپﻫﺎی اﯾﻤﻨﻮداﻣﯿﻨﻨﺖ ‪ HCV‬ ﭘﺎﺳﺦ اﯾﻤﻨﯽ ﻣﻨﺎﺳـﺐ‬ ‫ﻋﻠﯿﻪ آﻧﻬﺎ اﯾﺠﺎد ﺷﻮد.‬ ‫<br><b>روش ﺑﺮرﺳﯽ:</b> ﻗﻄﻌﺎت ژﻧﯽ ‪  HBsAg‬و ﭘﻨﺞ اﭘﯽ ﺗﻮپ ﻏﺎﻟﺐ ﺷﺎﻣﻞ core_132-142 , E2_405-414 , E2_614-622 , NS3_1406-1415 and core_39-48 ‬ﺑﻪ ﻗﻄﻌﻪ ژﻧﯽ ﺣﺎوی ﭘﺮوﻣﻮﺗﺮ  Hsp60‬و ‪  antigen signal sequence‬از ﻃﺮﯾﻖ ‪SOEing PCR‬‬ ‫ﻣﺘﺼﻞ، وﺗﮑﺜﯿﺮ ﺷﺪﻧﺪ. ﺳﭙﺲ ﻗﻄﻌﺎت ﺳﺎﺧﺘﻪ ﺷﺪه ﺷﺎﻣﻞ ﺗﺮادف ‪ HSP-S-HPOL‬در ﺷﺎﺗﻞ وﮐﺘﻮر  ‪  pVN2‬ﮐﻠﻮن‬ ‫ﺷﺪ. ﭘﻼﺳﻤﯿﺪ ﻧﻬﺎﯾﯽ(‪ (pHSP-alpha-S-HPOL‬ﺑﺎ روش اﻟﮑﺘﺮوﭘﻮرﯾﺸﻦ وارد ‪  BCG‬ﮔﺮدﯾﺪ. ‪  BCG‬ﻧﻮﺗﺮﮐﯿﺐ در ﻣﺤﯿﻂ‬ ‫ﮐﺸﺖ ﻟﻮﻧﺸﺘﺎﯾﻦ ﺟﺎﻧﺴﻮن ﺣﺎوی ﮐﺎﻧﺎﻣﺎﯾﺴﯿﻦ اﻧﮑﻮﺑﻪ ﺷﺪ.‬ ‫<br><b>ﯾﺎﻓﺘﻪ ﻫﺎ:</b> ﺳﺎزه ﻧﻬﺎﯾﯽ از ﻃﺮﯾﻖ ﻫﻀﻢ آﻧﺰﯾﻤﯽ و ﺗﻌﯿﯿﻦ ﺗﻮاﻟﯽ ﺗﺎﯾﯿﺪ ﺷﺪ. ﭘﺲ از ﮔﺬﺷﺖ 4 ﻫﻔﺘﻪ، ﮐﻠﻨﯽ ﻫﺎی ﻧﺨﻮدی رﻧﮓ‬ ‫وﻣﻘﺎوم ﺑﻪ ﮐﺎﻧﺎﻣﺎﯾﺴﯿﻦ ‪ BCG‬ ﻧﻮﺗﺮﮐﯿﺐ در ﺳﻄﺢ ﻣﺤﯿﻂ ﮐﺸﺖ ﻟﻮﻧﺸﺘﺎﯾﻦ ﺟﺎﻧﺴﻮن ﻧﻤﺎﯾﺎن ﺷﺪﻧﺪ. ﺗﺎﯾﯿﺪ ﭘﻼﺳﻤﯿﺪ در‬ ‫‪  BCG‬ﻧﻮﺗﺮﮐﯿﺐ ﺣﺎﺻﻞ ﺣﺎﮐﯽ از ﻣﻮﻓﻘﯿﺖ آﻣﯿﺰ ﺑﻮدن اﻟﮑﺘﺮوﭘﻮرﯾﺸﻦ ﺑﻮد.‬ <br>‫<b>ﻧﺘﯿﺠﻪﮔﯿﺮی: </b>ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد ﮐﻪ اﻣﮑﺎن ﺳﺎﺧﺖ ‪  rBCG‬ﻣﻮرد ﻧﻈﺮ ﭘﺲ از ﺳﺎﺧﺖ ﭘﻼﺳﻤﯿﺪ ﻧﻬﺎﯾﯽ ‪pHSP-S-HPOL‬‬ ‫و ﺗﺮاﻧﺴﻔﮑﺖ آن ﺑﻪ ‪ BCG‬ وﺟﻮد دارد. ﮐﻠﻨﯽﻫﺎی ‪  rBCG‬از ﻧﻈﺮ اﻣﮑﺎن ﺗﺮﺷﺢ ﭘﺮوﺗﺌﯿﻦ ﻧﻮﺗﺮﮐﯿﺐ ﻣﻮرد ﻧﻈﺮ ﻗﺎﺑﻞ ‫ﺑﺮرﺳﯽ ﻣﯽﺑﺎﺷﻨﺪ.‬ </div> <b>Background and objectives:</b> Liver diseases associated with chronic hepatitis c virus (HCV) infection are serious public health problems worldwide. There is neither an efficient therapy nor a vaccine available against this virus. Cellular immune responses, specially the cytotoxic T-lymphocytes (CTLs) play a key role towards the HCV clearance. Considering that the Bacillus Calmette-Guerin (BCG) as a tuberculosis vaccine has the ability to stimulate the cellular immunity, the final aim of this study was construction of a recombinant BCG (rBCG) as an HCV vaccine capable of secreting a polytope sequence composed of the immunodaminant HCV epitopes for the effective induction of HCV-specific CTLs. <br><b>Material and Method:</b> Hepatitis B surface antigen (HBsAg)-fused polytope sequence composed of 5 HCV epitopes derived from core_132-142 , E2_405-414 , E2_614-622 , NS3_1406-1415 and core_39-48 antigens made by SOEing PCR was further fused to the HSP60 promoter and BCG-alpha-antigen signal sequences. The produced HSP-S-HPOL fragment was further cloned into the kanamycin-encoding pVN2 shuttle vector and the final pHSPS-HPOL recombinant plasmid was transformed into the BCG cells by electroporation technique. The transformants were screened on the kanamycin containing media. <br><b>Results: </b>The pHSP-S-HPOL recombinant BCG-plasmid was successfully constructed and confirmed by restriction analyses and sequencing reactions. Transformation of BCG cells was efficiently achieved following intensive optimizations of electroporation parameters. The kanamycin-resistant transformants were screened in kanamycin-media in four weeks. <br> <b>Conclusion:</b> Appearance of kanamycin-resistant BCG colonies indicated the possibility of expression of pHSP-S-HPOL plasmid and its successful transformation, while motivated us to analyse the secretion of HCV-polytope and perform the in vivo immunogenicity assays. ﻫﭙﺎﺗﯿﺖ‪ ،c‬اﻟﮑﺘﺮوﭘﻮرﯾﺸﻦ ، ‪ BCG‬ﻧﻮﺗﺮﮐﯿﺐ‬ Hepatitis c, Electroporation, rBCG 18 28 http://ijmm.ir/browse.php?a_code=A-10-1-156&slc_lang=fa&sid=1 Attaollah Sadat Shandiz ﺳﯿﺪ ﻋﻄﺎاﻟﻪ ﺳﺎدات ﺷﺎﻧﺪﯾﺰ 1003194753284600941 1003194753284600941 No Biology Department, Khatam Institute of Higher Education, Tehran, Iran ﮔﺮوه زﯾﺴﺖ ﺷﻨﺎﺳﯽ، ﻣﻮﺳﺴﻪ آﻣﻮزش ﻋﺎﻟﯽ ﺧﺎﺗﻢ ﺗﻬﺮان Maryam Yazdanian ﻣﺮﯾﻢ ﯾﺰداﻧﯿﺎن 1003194753284600942 1003194753284600942 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran ﮔﺮوه ﻫﭙﺎﺗﯿﺖ واﯾﺪز، اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان Mohammadreza Aghasadeghi ﻣﺤﻤﺪ رﺿﺎ آﻗﺎﺻﺎدﻗﯽ 1003194753284600943 1003194753284600943 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran ﺑﺎﻧﮏ ژن ﻧﻮﺗﺮﮐﯿﺐ اﯾﺮان،اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان Hossein Khanahmad ﺣﺴﯿﻦ ﺧﺎن اﺣﻤﺪ 1003194753284600944 1003194753284600944 No BCG Department ,Research and Production Complex ,Pasteur Institute of Iran ﺑﺨﺶ ﺗﺤﻘﯿﻘﺎت ‪ ،BCG‬ﻣﺠﺘﻤﻊ ﺗﻮﻟﯿﺪی-ﺗﺤﻘﯿﻘﺎﺗﯽ اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان Arash Memarnejadian آرش ﻣﻌﻤﺎرﻧﮋادﯾﺎن 1003194753284600945 1003194753284600945 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran ﮔﺮوه ﻫﭙﺎﺗﯿﺖ واﯾﺪز، اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان Fatemeh Motevali ﻓﺎﻃﻤﻪ ﻣﺘﻮﻟﯽ 1003194753284600946 1003194753284600946 No NRGB Lab, Pasteur Institute of Iran, Tehran ﺑﺎﻧﮏ ژن ﻧﻮﺗﺮﮐﯿﺐ اﯾﺮان،اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان Tayebeh Sohrabi ﻃﯿﺒﻪ ﺳﻬﺮاﺑﯽ 1003194753284600947 1003194753284600947 No BCG Department ,Research and Production Complex ,Pasteur Institute of Iran ﺑﺨﺶ ﺗﺤﻘﯿﻘﺎت ‪ ،BCG‬ﻣﺠﺘﻤﻊ ﺗﻮﻟﯿﺪی-ﺗﺤﻘﯿﻘﺎﺗﯽ اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان Farzin Roohvand ﻓﺮزﯾﻦ روﺣﻮﻧﺪ rfarzin@pasteur.ac.ir 1003194753284600948 1003194753284600948 Yes Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran )NRGB Lab, Pasteur Institute of Iran, Tehran ﮔﺮوه ﻫﭙﺎﺗﯿﺖ واﯾﺪز، اﻧﺴﺘﯿﺘﻮ ﭘﺎﺳﺘﻮر اﯾﺮان