عنوان پایان‌نامه
تولید پروتئین نو ترکبیب هیرودین (Hirudin) بوسیله مخمر پروبیوتیک ساکارومایسس بولاردی
- رشته تحصیلی
- بهداشت و کنترل موادغذایی
- مقطع تحصیلی
- دکترای تخصصی
- محل دفاع
- کتابخانه دانشکده دامپزشکی شماره ثبت: 576 ت;کتابخانه دانشکده دامپزشکی - مخزن مرد آباد شماره ثبت: 576 ت;کتابخانه مرکزی -تالار اطلاع رسانی شماره ثبت: 64848
- تاریخ دفاع
- ۰۳ اردیبهشت ۱۳۹۳
- دانشجو
- حسن حامدی
- استاد راهنما
- علی میثاقی
- چکیده
- ساکارومایسس بولاردی بعنوان یک مخمر پروبیوتیک بخوبی شناخته شده و خواص درمانی و ارتقای سلامت زیادی به آن نسبت داده شده است. این مخمر شباهت زیادی به ساکارومایسس سرویزیه داشته ولی از نظر برخی خصوصیات تاکسونومی، متابویلیکی و ژنتیک تفاوتهایی دارند. این مخمر در نگهداری و احیای فلور طبیعی روده بزرگ و کوچک نقش دارد. در این پژوهش بااستفاده از UV موتاسیون با لامپ UV-C در فاصله 5/12 سانتی متری به مدت 22 ثانیه، سویه موتانت مخمر پروبیوتیک س. بولاردی بدست آمد. سویه آکسوتروف بدست آمده از نظر ژن URA3 با کشت انتخابی بر روی محیط انتخابی 5-FOA با مکمل های یوریدین/یوراسیل جداسازی و با کشت بر روی محیط YNB با مکمل های یوریدین/یوراسیل و بدون هیچگونه مکملی تایید گردید. این سویه با ترانسفورم کردن پلاسمید pYES2 که حاوی توالی ژن URA3 است با انجام آزمونهای کشت بر روی محیط انتخابی و PCR با پرایمرهای طراحی شده برای ژن URA3 تایید نهایی گردید. سپس آزمونهای پروبیوتیکی بر روی این سویه آکسوتروف مانند مقاومت به اسید و نمکهای صفراوی صورت گرفت و خواص پروبیوتیکی آن در مقایسه با سویه تجاری مورد تایید قرار گرفت. در ادامه طرح ، سازه بیانی داروی نوترکیب هیرودین که در بیماریهای قلبی عروقی کاربرد دارد، طراحی گردید. این سازه حاوی پروموتر TEF1 ، ?MF ، توالی داروی نوترکیب هیرودین (HV2-K47) و سایت های برش آنزیمی بود. این توالی بعد از سنتز و تایید با هضم آنزیمی با پلاسمید pYES2 ، ligate گردید. این سازه نهایی با استفاده از الکتروپوریشن به داخل سویه آکسوتروف س. بولاردی ترانسفورم گردید. تایید ترانسفورم با استخراج پلاسمید از مخمر و انجام هضم آنزیمی و PCR با پرایمرهای طراحی شده برای سازه بیانی هیرودین صورت گرفت. در مرحله بعدی بعد از کشت مخمر ترانسفورم شده با سازه بیانی، با انجام SDS-PAGE و جداسازی پروتئین هیرودین و انجام وسترن با آنتی بادی اختصاصی مونوکلنال هیرودین و همچنین با کیت الایزا بیان ژن هیرودین در این مخمر پروبیوتیکی تایید گردید. تایید بیان ژنی هیرودین با استفاده از روش جداسازی cDNA و انجام RT-PCR نیز انجام گرفت. نتایج بدست آمده از این پژوهش یک سویه نوترکیب از مخمر پروبیوتیکی است که حاوی ژن هیرودین بوده و قابلیت ترشح این داروی نوترکیب در داخل دستگاه گوارش میزبان را دارد.
- Abstract
- Saccharomyces boulardii is well known as a probiotic yeast and many health-promoting properties has been attributed to it. It is related to, but distinct from, Saccharomyces cerevisiae in several taxonomic, metabolic, and genetic properties. S. boulardii has been shown to maintain and restore the natural flora in the large and small intestine. In this study, in order to generate auxotrophic mutants, cells were placed under a UV lamp at a distance of 12.5 cm and were irradiated for 22 seconds. Auxotrophic mutants were screened by plating method on 5-FOA selective medium with necessary supplements, uridine and uracil. Confirmation of these isolated strains was performed with grown on a minimal medium (YNB medium with and without uridine and uracil supplements). To confirm the Ura3 gene disruption in uridine auxotroph mutants, a selected mutant was transformed with pYES2 plasmid containing Ura3 gene sequence. The positive transformants were isolated on minimal medium lacking uridine and uracil. Further confirmation was performed by isolation of pYES2 from ura3+ transformants. The probiotic properties of the auxotrophic mutants such as resistance to acid and bile salts in comparison with the commercial probiotic strain were approved. An expression construct comprising TEF1 promoter, hirudin sequence and the termination signal, CYC1 was prepared in pYES2 plasmid. In this way, the original Gal1 promoter in pYES2 was replaced by TEF1 promoter to drive a constitutive expression of hirudin. This construct subsequently was transformed in Saccharomyces boulardii Ura3- strain. The Ura+ transformants were selected on Minimal medium lacking uridine and uracil. Further confirmation of positive transformants containing the expression cassette was performed through plasmid extraction from these transformants and restriction mapping of extracted plasmids. Subsequently, the recombinant strains were checked for the expression of hirudin. As the designed construct contains a signal sequence to support the secretion of recombinant hirudin to the culture medium, we tested the presence of rHirudin in culture medium by ELISA Kit, Western Blot with monoclonal antibody and RT-PCR with isolated cDNA. In conclusion, we have generated a recombinant probiotic yeast strain that is capable of producing recombinant hirudin .This strain may release the sufficient amounts of hirudin inside the host's gastrointestinal tract to prevent abnormal blood coagulation. Saccharomyces boulardii is well known as a probiotic yeast and many health-promoting properties has been attributed to it. It is related to, but distinct from, Saccharomyces cerevisiae in several taxonomic, metabolic, and genetic properties. S. boulardii has been shown to maintain and restore the natural flora in the large and small intestine. In this study, in order to generate auxotrophic mutants, cells were placed under a UV lamp at a distance of 12.5 cm and were irradiated for 22 seconds. Auxotrophic mutants were screened by plating method on 5-FOA selective medium with necessary supplements, uridine and uracil. Confirmation of these isolated strains was performed with grown on a minimal medium (YNB medium with and without uridine and uracil supplements). Saccharomyces boulardii is well known as a probiotic yeast and many health-promoting properties has been attributed to it. It is related to, but distinct from, Saccharomyces cerevisiae in several taxonomic, metabolic, and genetic properties. S. boulardii has been shown to maintain and restore the natural flora in the large and small intestine. In this study, in order to generate auxotrophic mutants, cells were placed under a UV lamp at a distance of 12.5 cm and were irradiated for 22 seconds. Auxotrophic mutants were screened by plating method on 5-FOA selective medium with necessary supplements, uridine and uracil. Confirmation of these isolated strains was performed with grown on a minimal medium (YNB medium with and without uridine and uracil supplements). To confirm the Ura3 gene disruption in uridine auxotroph mutants, a selected mutant was transformed with pYES2 plasmid containing Ura3 gene sequence. The positive transformants were isolated on minimal medium lacking uridine and uracil. Further confirmation was performed by isolation of pYES2 from ura3+ transformants. The probiotic properties of the auxotrophic mutants such as resistance to acid and bile salts in comparison with the commercial probiotic strain were approved. An expression construct comprising TEF1 promoter, hirudin sequence and the termination signal, CYC1 was prepared in pYES2 plasmid. In this way, the original Gal1 promoter in pYES2 was replaced by TEF1 promoter to drive a constitutive expression of hirudin. This construct subsequently was transformed in Saccharomyces boulardii Ura3- strain. The Ura+ transformants were selected on Minimal medium lacking uridine and uracil. Further confirmation of positive transformants containing the expression cassette was performed through plasmid extraction from these transformants and restriction mapping of extracted plasmids. Subsequently, the recombinant strains were checked for the expression of hirudin. As the designed construct contains a signal sequence to support the secretion of recombinant hirudin to the culture medium, we tested the presence of rHirudin in culture medium by ELISA Kit, Western Blot with monoclonal antibody and RT-PCR with isolated cDNA. In conclusion, we have generated a recombinant probiotic yeast strain that is capable of producing recombinant hirudin .This strain may release the sufficient amounts of hirudin inside the host's gastrointestinal tract to prevent abnormal blood coagulation.
