طراحی و ساخت یک پلاسمید عرضه سطحی حاوی قطعه ژنی رمزگذار پروتئین E6 نوترکیب ویروس پاپیلومای انسانی تیپ 18 جهت بیان در مخمر Yarrowia lipolytica

نوع مقاله : اصیل پژوهشی

نویسندگان

1 کارشناس ارشد زیست‌شناسی سلولی و مولکولی، دانشکده علوم پایه، دانشگاه شهید مدنی آذربایجان، تبریز، ایران.

2 استادیار گروه زیست‌شناسی سلولی و مولکولی، دانشکده علوم پایه، دانشگاه شهید مدنی آذربایجان، تبریز، ایران.

3 استاد گروه میکروبیولوژی، دانشکده علوم زیستی، دانشگاه الزهراء، تهران، ایران.

4 محقق بالارتبه، انستیتو تحقیقات ملی کشاورزی، غذا و محیط، دانشگاه Prais-Saclay، پاریس، فرانسه.

چکیده

مقدمه: ویروس­ های پاپیلومای انسانی (HPV) با بیش از 100 تیپ، به دو دسته­ با ریسک پایین و بالا تقسیم می­ شوند که تیپ­ های 16 و 18 به‌تنهایی در 70% موارد سرطان گردنه­ رحم دخیل­ اند. در حال حاضر، توسعه­ پروتئین ­های نوترکیب HPV با هدف واکسیناسیون یا درمان مورد توجه دانشمندان می‌باشد. از این رو، مطالعه حاضر با هدف ساخت یک پلاسمید نوترکیب عرضه‌کننده در سطح رمزگذار پروتئین E6 ویروس پاپیلومای انسانی تیپ 18 انجام شد.
روش‌کار: قطعه­ ژنی رمزگذار پروتئین E6 تیپ 18 ویروس پاپیلومای انسانی (HPV18) با استفاده از DNA فرد مبتلا به ویروس به‌عنوان الگو، با روش PCR آشیانه­ ای مورد تکثیر قرار گرفت و پس از برش آنزیمی دوگانه­ Hind III و SfiI، به درون پلاسمید عرضه­ کننده در سطح pINA1317-YLCWP110 همسانه­ سازی شد.
یافته ­ها: روش ­های مولکولی نظیر PCR و برش آنزیمی دوگانه صحت همسانه­ سازی پلاسمید­ نوترکیب pINA1317-YLCWP110-E6 را مورد تأیید قرار داد. همچنین، نتایج به‌دست آمده از تعیین توالی به‌روش سنگر و هم­ردیفی با داده­ های موجود در بانک ژن منجر به تأیید نهایی صحت توالی، ترادف و چارچوب قرار­ گرفتن قطعه­­ ژنی در جایگاه مناسب گردید.
نتیجه­ گیری: نتایج این مطالعه، همسانه­ سازی موفق قطعه­ ژنی رمزگذار پروتئین E6 ویروس HPV18 را در پلاسمید عرضه کننده در سطح pINA1317-YLCWP110-E6 در جایگاه و جهت مناسب مورد تأیید قرار داد. این پلاسمید در صورت بیان در میزبان مخمری Yarrowia lipolytica، قابلیت استفاده به‌عنوان واکسن، مارکر مولکولی یا جنبه­ درمانی خواهد داشت. 

کلیدواژه‌ها

عنوان مقاله [English]

Developing a plasmid for surface display containing the recombinant E6 protein of human papilloma virus type 18 for expression in yeast Yarrowia lipolytica

نویسندگان [English]

  • Mohaddeseh Koohichapan 1
  • Solmaz Moniri Javadhesari 2
  • Farshad Darvishi Harzevili 3
  • Catherine Madzak 4
1 M.Sc. of Cellular and Molecular Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran.
2 Assistant Professor, Department of Cellular and Molecular Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran.
3 Professor, Department of Microbiology, School of Biological Sciences, Alzahra University, Tehran, Iran.
4 INRAE France’s New National Research Institute for Agriculture, Food and Environment, Paris-Saclay University, Paris, France.
چکیده [English]

Introduction: Human papillomaviruses (HPV) with more than 100 types are categorized as low-risk and high-risk types. Types 16 and 18 of the virus alone are involved in 70% of cervical cancers. Currently, development of recombinant proteins of HPV for vaccination or therapy purposes has attracted scientists. Therefore, the present study was performed aimed to construct a surface display plasmid encoding E6 protein of human papillomavirus type 18.
Methods: The DNA fragment encoding E6 protein of HPV18 was amplified by nested-PCR using DNA of a HPV18 positive person as PCR template. Then, the amplified fragment was double digested with HindIII and SfiI and cloned into the surface display plasmid pINA1317-YLCWP110.
Results: Cloning of E6 protein encoding gene fragment into pINA1317-YLCWP110 plasmid was confirmed using PCR and restriction endonuclease double digestion. Also, the results of Sanger sequencing of the recombinant pINA1317-YLCWP110-E6 plasmid and alignment to gene bank further ensured the sequence accuracy, cloning position and reading frame of the gene in the recombinant vector.
Conclusion: DNA fragment encoding E6 protein of HPV18 was successfully cloned into surface display plasmid pINA1317-YLCWP110 in appropriate locus and frame. Altogether, the recombinant pINA1317-YLCWP110-E6 plasmid constructed in this study can be expressed in the yeast host Yarrowia lipolytica and the resulted E6 protein may be used as a prophylactic or therapeutic vaccine or molecular marker.

کلیدواژه‌ها [English]

  • Recombinant E6 protein
  • Surface display plasmid
  • Type 18
  • Human Papilloma Virus
  • Cloning

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مجله زنان، مامایی و نازایی ایران
دوره 25، شماره 8
آبان 1401
صفحه 68-80

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