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مهندسی یک پروتئین کایمریک نوترکیب متشکل از آنتیژن OmpL متصل شده به ادجوانت مولکولی HBHA (HBHA-OMPL) با هدف توسعه واکسن بر علیه سالمونلا تیفی موریوم | ||
| تحقیقات دامپزشکی و فرآوردههای بیولوژیک | ||
| مقاله 12، دوره 36، شماره 2، تیر 1402، صفحه 100-111 اصل مقاله (3.08 M) | ||
| نوع مقاله: مقاله کامل | ||
| شناسه دیجیتال (DOI): 10.22092/vj.2022.359453.1995 | ||
| نویسندگان | ||
| مرضیه بازگیر1؛ نعمت شمس* 2؛ علی فروهرمهر3؛ امین جایدری4؛ نرگس نظیفی2 | ||
| 1گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه لرستان,، ایران | ||
| 2گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه لرستان، ایران | ||
| 3گروه علوم دام، دانشکده کشاورزی، دانشگاه لرستان، ایران | ||
| 4گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه لرستان، ایران | ||
| چکیده | ||
| سالمونلا تیفی موریوم یک باکتری گرم منفی از خانواده آنتروباکتریاسه است که بعنوان عامل ایجاد کننده بیماری در انسان و دام مطرح میباشد. یکی از جدیدترین راه های مقابله با این عامل بیماریزا استفاده از واکسنهای نوترکیب است. در این مطالعه، سازهای متشکل از آنتیژن OmpL از باکتری سالمونلا تیفــیموریــوم و پروتئین HBHA به عنوان ادجوانت مولکولی از باکتری مایکوباکتریوم به روش درون رایانه ای (in silico) و با استفاده از یک لینکر پپتیدی طراحی شد. بدین منظور توالی نوکلئوتیدی هریک از این پروتئینها از پایگاه دادهها استخراج شدند و سازه نوترکیب HBHA-OmpL با بررسی فریم صحیح خوانش طراحی شد. سپس برای ارزیابی امتیاز ایمنیزایی و خواص فیزیکوشیمیایی و همچنین ساختارهای دوم و سوم این سازه از سرورهای معتبر آنلاین استفاده شد. بهترین مدل سه بعدی پالایش شده برای فرایند داکینگ مولکولی مورد استفاده قرار گرفت. توالی نکلئوتیدی سازه کایمریک نوترکیب برای بیان در اشریشیا کولای بهینه سازی گردید و امکان کلون توالی بهینه شده در وکتور بیانی (+)pET22b بررسی شد. بر اساس نتایج بدست آمده در این مطالعه، سازه کایمریک HBHA- OmpL با وزن مولکولی 45/49 کیلو دالتون دارای شاخص ناپایداری 75/32 و شاخص آنتی ژنسیتی 688/0 بود و ساختارهای مارپیچ آلفا و سیم پیچهای تصادفی بیشترین سهم را در توزیع ساختار دوم این سازه داشتند. بررسی داکینگ پروتئین-پروتئین نشان داد که پروتئین HBHA علیرغم کانژوگه شدن با پروتئین OmpL، توانست از طریق 23 پیوند هیدروژنی به گیرنده MD2/TLR4متصل شود. نتایج کلونینگ درون رایانه ای نیز نشان داد سازه کایمریک میتواند در وکتور (+)pET22b با موفقیت کلون شود. | ||
| کلیدواژهها | ||
| واکسن نوترکیب؛ سالمونلا تیفیموریوم؛ OmpL؛ HBHA | ||
| عنوان مقاله [English] | ||
| Engineering a Recombinant Chimeric Protein Consisting of OmpL Antigen Conjugated to HBHA Molecular Adjuvant (HBHA-OmpL) with the Aim of Developing a Vaccine Against Salmonella Typhimurium | ||
| نویسندگان [English] | ||
| Marzieh Bazgir1؛ Nemat Shams2؛ Ali Forouharmehr3؛ Amin Jaydari4؛ Narges Nazifi2 | ||
| 1,,Department of Pathobiology,Faculty of Veterinary Medicine, Lorestan University, Iran | ||
| 2Department of Pathobiology, Faculty of Veterinary Medicine, Lorestan University, Iran | ||
| 3Department of Animal Science, Faculty of Agriculture, Lorestan University, Iran | ||
| 4Department of Pathobiology, Faculty of Veterinary Medicine, Lorestan University, Iran | ||
| چکیده [English] | ||
| Salmonella Typhimurium is a zoonotic gram-negative bacterium of the Enterobacteriaceae family. One of the newest ways against the pathogen is the use of recombinant vaccines. In this study, a recombinant construct consisting of OmpL antigen from Salmonella Typhimurium and HBHA protein as a molecular adjuvant from Mycobacterium was designed by an in silico method and a peptide linker. For this purpose, sequences of these proteins were extracted from the database and the HBHA-OmpL recombinant construct was designed based on the correct reading frame. Then, reliable online servers were used to evaluate the immunogenicity score and physicochemical properties as well as the secondary and tertiary structures of the designed construct. The best refined 3D model was used for the molecular docking process. A codon optimization was done for expression in E. coli, and the possible cloning of the optimized nucleotide sequence in pET22b(+) expression vector was evaluated. Based on the results obtained in this study, the HBHA-OmpL chimeric construct with a molecular weight of 49.45 kDa had an instability index of 32.75 and an antigenicity index of 0.688, and alpha helices and random coil structures had the largest contribution in its secondary structure. The protein-protein docking results showed that despite being conjugated with OmpL protein, HBHA molecule was able to connect to TLR4/MD2 receptor by 23 hydrogen bonds. Also, cloning results confirmed that nucleotide sequence of the designed vaccine can be successfully inserted in pET22b(+) vector. | ||
| کلیدواژهها [English] | ||
| Recombinant vaccine, Salmonella typhimurium, OmpL, HBHA | ||
| مراجع | ||
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