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پژوهش‌های آسیب‌ شناسی زیستی، جلد ۱۸، شماره ۲، صفحات ۲۵-۳۴
عنوان فارسی بررسی تأثیر استفاده از پپتید نشانه پروترومبین انسانی بر کارآیی ترشح فاکتور۹ انسانی در رده سلولی HEK۲۹۳T
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عنوان انگلیسی Studying the effect of human prothrombin’s signal peptide on the secretion efficiency of recombinant human FIX in HEK293T cell line
چکیده انگلیسی مقاله Background: Eukaryotic proteins generally have signal peptides, which are not only crucial for their secretion efficiencies but are important for their expression levels. The coagulation factor IX (FIX) is a glycoprotein that plays a fundamental role in a blood coagulation pathway. Reduced levels or dysfunctional FIX are associated with hemophilia B. To improve the hFIX secretion efficiency in a heterologous expression system, in this study we investigated the function of the human prothrombin signal peptide. With this aim, we used SignalP, and PrediSi programs for in silico evaluation of the signal peptides efficiency before being examined experimentally. Methods: Using molecular techniques, we amplified and joined the coding region of the human prothrombin signal peptide to the cDNA of mature hFIX. The chimeric fragment was examined for transient expression in mammalian cell line (HEK293T) in comparison with the native hFIX, under a CMVp regulation. Using the neural network-based prediction programs, we evaluated the scores for cleavage position and secretion efficiency of the human prothrombin and hFIX signal peptides. The expression efficiencies of hFIX expressed by the recombinant cells was analyzed by RT-PCR, ELISA. Results: In silico analysis predicted the human prothrombin signal peptide with the high score more efficient than the native hFIX signal peptide. These data was confirmed by the results obtained from the expression analysis in RNA and protein level of the rhFIX by RT-PCR and ELISA, respectively. Conclusion: The present study showed that, the signal peptide derived from the human prothrombin has the potential for efficient secretion of hFIX, which was evidenced by the results taken from a transient expression system and it was in consistent with in silico analysis. This replacement can be evaluated in stable state.
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نویسندگان مقاله شهره خورشیدی | shohreh khorshidi
department of genetics, faculty of biological sciences, tarbiat modares university, tehran, iran
سازمان اصلی تایید شده: دانشگاه تربیت مدرس (Tarbiat modares university)

علیرضا زمردی پور | alireza zomorodipour
department of molecular medicine, institute of medical biotechnology, national institute of genetic engineering and biotechnology, tehran, iran
سازمان اصلی تایید شده: دانشگاه تربیت مدرس (Tarbiat modares university)

مهرداد بهمنش | mehrdad behmanesh
department of genetics, faculty of biological sciences, tarbiat modares university, tehran, iran
سازمان اصلی تایید شده: پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری

نشانی اینترنتی http://mjms.modares.ac.ir/article_13032_a113e8f0d135ef1dc17198372e277fe1.pdf
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