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High-level Expression of Tetanus Toxin Fragment C in Escherichia coli | ||
| Archives of Razi Institute | ||
| مقاله 4، دوره 73، شماره 1، خرداد 2018، صفحه 27-38 اصل مقاله (506.71 K) | ||
| نوع مقاله: Original Articles | ||
| شناسه دیجیتال (DOI): 10.22092/ari.2018.114057 | ||
| نویسندگان | ||
| K. Aghaiypour* ؛ R. Teymourpour | ||
| Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Agricultural Research, Education, and Extension Organization, Karaj, Iran | ||
| چکیده | ||
| Fragment C is the C-terminal domain of the heavy chain of tetanus toxin that can promote the immune response against the lethal dose of this toxin. Therefore, this portion can be considered as a candidate vaccine against tetanus infection, which occurs by Clostridium tetani. The present study aimed to compare the expression of tetanus toxin fragment C in Escherichia coli BL21 (DE3) pLysS cells having a high tolerance to toxins between two different expression vectors, namely pET22b and pET28a, using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. After DNA extraction from Harvard CN49205 strain of C. tetani, the gene of interest was amplified using polymerase chain reaction, and then sequenced and cloned into the expression vectors of pET22b and pET28a, transformed into competent BL21 (DE3) pLysS cells, and finally expressed using an optimized protocol. The cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at four different incubation temperatures (i.e., 37, 33, 30, and 25 °C) and three different incubation times (i.e., 1, 2, and 3 h). Although the SDS-PAGE and western blot analyses confirmed the expression of the recombinant fragment C (r-fragment C) ligated into both of the expression vectors, pET28a showed a higher r-fragment C expression level than the other vector (38.66 mg/L versus 32.33 mg/L, P<0.05). An optimal expression condition was acquired 3 h after 1 mM IPTG induction at 25 °C. The results demonstrated that E. coli BL21 (DE3) pLysS as an expression host in combination with pET-28a as an expression vector was a more compatible expression system to express the fragment C of tetanus toxin, compared to E. coli BL21 (DE3) pLysS/pET-22b expression system. Overall, these results may represent an opportunity to improve the expression system for the production of tetanus toxin vaccine using recombinant protein strategy. | ||
| کلیدواژه‌ها | ||
| Clostridium tetani؛ Fragment C؛ pET22b expression vector؛ pET28a expression vector؛ E. coli BL21 (DE3) pLysS | ||
| عنوان مقاله [English] | ||
| بیان بسیار بالای قطعه C سم تتانوس در اشرشیا کلی | ||
| نویسندگان [English] | ||
| خسرو آقائی پور؛ رقیه تیمورپور | ||
| بخش ژنومیکس و مهندسی ژنتیک، مدیریت بیوتکنولوژی و آزمایشگاه مرکزی، موسسه تحقیقات واکسن و سرم سازی رازی، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران | ||
| چکیده [English] | ||
| قطعه C، انتهای کربوکسیل زنجیره سنگین سم تتانوس میباشد که میتواند پاسخ ایمنی علیه دوز کشنده این سم را تحریک نماید. این پلی پپتید میتواند به عنوان کاندیدای واکسن علیه عفونت تتانوس که توسط باکتری کلستریدیوم تتانی ایجاد میشود مطرح شود. این مطالعه بر روی مقایسه بیان قطعه C سم تتانوس در سلول BL21(DE3)pLysS که تحمل بالایی به سم تتانوس دارد با دو وکتور pET22b و pET28a به کمک آنالیزسدیم دودسیل سولفات پلی آکریل آمید ژل و وسترن بلات انجام شد. پس از استخراج DNA سویه هاروارد CN49205 کلستریدیوم تتانی، ژن هدف بکمک واکنش زنجیری پلیمراز تکثیر و سپس تعیین توالی شده و درون وکتورهای pET22b و pET28a کلون شد. سپس وکتورها داخل سلول BL21(DE3)pLysS تانسفورم شد و سپس توسط یک پروتکل از پیش بهینه شده بیان شد. سلولها توسط ایزوپروپیل بتا دی-1- تیو گالاکتوپیرانوزید (IPTG) در چهار تیمار 25، 30، 33 و 37 درجه سانتیگراد و سه زمان متفاوت 1، 2 و 3 ساعت بیان شد. اگرچهSDS-PAGE و وسترن بلات بیان قطعه C نوترکیب (r-fragment C) وارد شده به هر دو وکتور را تایید کرد اما وکتور pET28a نشان دهنده بیان بسیار بالاتر ( 66/38 میلی گرم بر لیتر در مفایسه با 33/32 میلی گرم بر لیتر pET22b بود (< 0.05 P). شرایط بیان بهینه پس از 3 ساعت تیمار با mM IPTG در 25 درجه سانتیگراد بود. نتایج نشان داد که وکتور pET28a و سلول BL21(DE3)pLysS تواناتر و سازگارتر برای بیان قطعه C سم تتانوس در مقایسه با وکتور pET22b و BL21(DE3)pLysS بود. این نتایج نگرشهای جدیدی برای بیان پروتئینهای سمی چون فرگمنت C به کمک فناوری نوترکیب در اشرشیا کلی را ارائه مینماید. | ||
| کلیدواژه‌ها [English] | ||
| کلستریدیوم تتانی, قطعه C, وکتور بیانی pET22b, وکتور بیانی pET28a, اشریشیا کلیBL21 (DE3) pLysS | ||
| مراجع | ||
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