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Development of a polyclonal antibody against the coat protein of Prunus necrotic ring spot virus | ||
| Iranian Journal of Genetics and Plant Breeding | ||
| دوره 12، شماره 2 - شماره پیاپی 24، دی 2023، صفحه 61-70 اصل مقاله (803.45 K) | ||
| نوع مقاله: Research paper | ||
| شناسه دیجیتال (DOI): 10.30479/ijgpb.2024.19400.1357 | ||
| نویسندگان | ||
| Sara Ebrahimi؛ Davoud Koolivand* | ||
| Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran. | ||
| تاریخ دریافت: 15 مهر 1402، تاریخ بازنگری: 20 فروردین 1403، تاریخ پذیرش: 21 فروردین 1403 | ||
| چکیده | ||
| The Prunus necrotic ring spot virus (PNRSV) poses a significant threat to the global stone fruit industry. In this study, samples suspected of PNRSV infection were gathered from Iran’s primary stone fruit-growing areas. The coat protein (CP) gene of the isolates was amplified, and the nucleic acid and amino acid sequences of the PNRSVs were determined. Phylogenetic analysis revealed that these isolates belonged to the PV-96-II, a prominent group of PNRSVs found worldwide. An isolate from this clade was chosen to express the CP gene in Escherichia coli. The CP gene of this isolate was cloned into the pET28 vector for expression, and the resulting coat protein was purified as a native protein using Ni-NTA agarose. The purified protein served as a recombinant antigen to generate anti-PNRSV-CP antiserum in rabbits. Purified anti-PNRSV-CP IgG and conjugated IgG showed specificity and sensitivity, successfully detecting expressed CP and PNRSV isolates in infected stone fruit trees through various serological and sero-molecular techniques such as plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), Double Antibody Sandwich-ELISA (DAS-ELISA), and western blotting. These antibodies can be valuable in virus and plant screening studies, as well as serological and sero-molecular tests. This study represents the first documentation of a polyclonal antibody preparation against the CP of an Iranian PNRSV isolate, offering potential assistance in controlling and preventing this economically significant virus in the future. | ||
| کلیدواژه‌ها | ||
| Antibody؛ Competent cell؛ Purification؛ Polymerase chain reaction؛ Prokaryotic system | ||
| عنوان مقاله [English] | ||
| تولید آنتی‌بادی چند هم‌سانه‌ای علیه پروتئین پوششی ویروس لکه حلقوی نکروتیک هسته‌داران | ||
| نویسندگان [English] | ||
| سارا ابراهیمی؛ داود کولیوند | ||
| گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه زنجان، زنجان، ایران. | ||
| چکیده [English] | ||
| ویروس لکه حلقوی نکروتیک هسته‌داران (Prunus necrotic ring spot virus)، تهدید مهم و قابل توجهی برای درختان هسته‌دار در دنیا است. در این مطالعه، نمونه‌های مشکوک به آلودگی PNRSV از مناطق مهم و اصلی کاشت درختان هسته‌دار در ایران، جمع‌آوری شدند. ژن پروتئین پوششی (CP) جدایه‌ها در آزمون پی‌سی‌آر تکثیر شد و قطعات تکثیر یافته، تعیین توالی شدند. تجزیة تحلیل تبارزایی نشان داد که این جدایه‌ها، به گروه PV-96-II تعلّق دارند که یک گروه مهم از PNRSVها در سراسر جهان هستند. یک جدایه از این گروه، برای بیان ژن پروتئین پوششی در E. coli انتخاب شد. ژن CP این جدایه، به وکتور pET28 برای بیان هم‌سانه‌سازی شد و پروتئین پوششی بیان شده با روش Native با استفاده از ستون Ni-NTA خالص‌سازی شد. پروتئین خالص شده، به‌عنوان یک آنتی‌ژن برای تولید آنتی سرم، علیه پروتئین پوششی PNRSV در خرگوش‌ها استفاده شد. IgG تولید شده علیه PNRSV-CP تخلیص شده و IgG کانژوگه شده، حساسیت و اختصاصیت لازم را نشان دادند و با موفقیت CP بیان شده و جدایه‌های PNRSV را در درختان میوه‌های هسته‌دار آلوده، از طریق تکنیک‌های مختلف سرومولکولی و سروولوژیکی؛ ازجمله آزمون PTA-ELISA، DAS-ELISA و وسترن بلات ردیابی شدند. این آنتی‌بادی‌ها می‌توانند در مطالعات ردیابی ویروس در گیاه و همچنین آزمایش‌های سروولوژیکی و سرومولکولی قابل استفاده باشند. این مطالعه، نخستین مطالعه‌ای است که در مورد تهیة آنتی‌بادی چندهمسانه‌ای، در برابر CP جدایة PNRSV ایرانی صورت گرفته است و در کنترل و پیش‌گیری از این ویروس مهم اقتصادی، در آینده کمک خواهند نمود. | ||
| کلیدواژه‌ها [English] | ||
| آنتی‌بادی, سلول مستعد, خالص‌سازی, واکنش زنجیره‌ای پلی‌مزاز, سیستم پروکاریوتی | ||
| مراجع | ||
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