2دانشگاه تربیت مدرس، دانشکده کشاورزی، تخصص: بیماری های طیور
3عضو هیات علمی پژوهشگاه رویان
4عضو هیات علمی گروه علوم طیور، دانشگاه تربیت مدرس
چکیده
هدف از این مطالعه، همسانه‌سازی‌زیرواحد بتای هورمون گونادوتروپین جفت انسانی در ناقل مناسب بود که بتواند از طریق اسپرم در تولید طیور تراریخت به کار گرفته شود. به این منظور زیرواحد بتای این هورمون به کمک یک جفت آغازگر اختصاصی تکثیر و درناقل T همسانه‌سازی شد. فرآیند ترانسفورماسیون پلاسمید نوترکیب در سلول‌های مستعد اشریشیاکلی انجام گرفت و کلونی‌های دارای پلاسمید نوترکیب به‌وسیله PCR انتخاب شدند. صحت تخلیص پلاسمید ابتدا توسط هضم آنزیمی و نهایتاً به روش توالی‌یابی بررسی شد. پس از آن، زنجیره بتا از ناقلT جداسازی شد و مجددا در ناقل بیانی pcDNA3.1+ همسانه‌سازی شد. نتایج آنالیز آنزیمی و تعیین توالی نشان داد که پلاسمید نوترکیب hCGβ/ pCDNA3.1+ با توالی صحیح ساخته شد و تطابق کامل با ژن زیرواحد بتای هورمون گونادوتروپین جفت انسانی داشت. می توان نتیجه گرفت که پلاسمید نوترکیب حاوی زیر واحد بتای گونادوتروپین جفت انسانی ساختار مناسبی برای انتقال به اسپرم خروس دارد که می تواند در تولید جوجه های تراریخت به کار گرفته شود.
Cloning of human chorionic gonadotropin beta subunit for rooster sperm mediated gene transfer
نویسندگان [English]
Mehdi Noorani1؛ Shaban Rahimi2؛ Abdolhoseein Shahverdi3؛ Mohsen Sharafi4
1MSc in Poultry Science, Tarbiat Modares University
2Professor Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University
3Faculty member at Royan Institute
4Faculty member at the Department of Poultry Science, Tarbiat Modares University
چکیده [English]
The aim of this study was cloning of the beta subunit of human chorionic gonadotropin in an appropriate vector for production of transgenic chicken trrough sperm mediated gene transfer. In this regard, transgenic chicken production tecnology has taken into consideration for having many advantages such as short generation times, the large number of production of offspring and suitable pattern of protein glycosylation. To date, no study has been conducted on the cloning of the beta subunit of human chorionic gonadotropin for rooster sperm. For this purpose, the hormone beta subunit were amplified by a specific primer pairs, and cloned in T vector. The recombinant plasmid was transformed into Competent E. coli cells and colonies that containing recombinant plasmids were selected by colony PCR.The validity of extracted plasmid were analyzed by enzyme digestion and sequencing. The beta chain of T vector was isolated and was cloned again into pcDNA3.1 + expression vector. The results of enzyme analysis and sequencing indicated that recombinant plasmid pCDNA3.1 +/βhCG were cloned with the correct sequence and completely matched up with human chorionic gonadotropin beta subunit gene that can be concluded that it has sutible stracture for sperm mediated gene transfer.
کلیدواژه‌ها [English]
plasmid, sperm, sequencing, transgenic chicken
مراجع
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