زیر همسانه سازی و بیان ژن SO9 گیاه غاسول صابونی در باکتری E. coli و بررسی تیتر آنتی بادی آن در موش سوری

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد بیوتکنولوژی گیاهی، مرکز علوم و تکنولوژی، دانشگاه پیام نور، تهران، ایران

2 دانشیار علوم کشاورزی، مرکز علوم و تکنولوژی، دانشگاه پیام نو، تهران، ایران

3 استاد سیستماتیک گیاهی، مرکز علوم و تکنولوژی، دانشگاه پیام نور، تهران، ایران

4 دانشجوی دکتری باکتری‌شناسی، مرکز علم و فناوری زیست‌شناسی، دانشکده علوم پایه، دانشگاه جامع امام حسین(ع)، تهران، ایران

چکیده

: غاسول صابونی دارای ایزوفرم های مختلف پروتئین ساپورین می باشد. ساپورین جزء پروتئین های غیر فعال کننده ریبوزومی (RIPs) است. ایزوفرم SO9 ساپورین ها، آدنین 4324 موجود در توالی حفاظت شده GAGA را دپورینه کرده و باعث اختلال در پروتئین سازی می‌گردد. در این مطالعه بیان ایزوفرم SO9 در باکتری E. coli و بررسی تیتر آنتی بادی آن در موش سوری انجام شد.
روش بررسی: ژن SO9 سنتز شده، از پلاسمید pUC57-SO9 نوترکیب توسط آنزیم های محدود کننده BamH1 و Sal1 جدا و در وکتور بیانی pET28a(+) زیر همسانه سازی شد. بیان پروتئین نوترکیب با IPTG القا گردید. پروتئین SO9 نوترکیب به وسیله کروماتوگرافی تمایلی نیکل خالص سازی شد. برای تایید پروتئین نوترکیب از تکنیک Western blotting استفاده شد. واکسیناسیون موش های سوری با پروتئین تخلیص شده به صورت صفاقی انجام و تیتر IgG سرم به وسیله ELISA بررسی شد.
نتایج: زیر همسانه سازی ژن SO9 در وکتور بیانی pET28a(+) به وسیله واکنش PCR و هضم آنزیمی تایید شد. حضور باند پروتئینی 29 کیلودالتون در SDS-PAGE، بیان بالای پروتئین نوترکیب را نشان داد. پروتئین نوترکیب SO9 به وسیله آنتی بادی پلی کلونال شناسایی شد. بعد از تزریق پروتئین در گروه های تست نسبت به گروه های کنترل، میزان تیتر آنتی بادی تولید شده به وسیله تست ELISA اندازه گیری شد.
نتیجه گیری: خاصیت ادجوانتی و ایمنوژنی آنتی ژن SO9 نوترکیب تخلیص شده سبب می‌شود که از این آنتی بادی برای شناسایی میزان حضور SO9 در گیاه غاسول صابونی، به عنوان کاندید واکسن، تهیه کیت تشخیصی و در مطالعات ضد سرطانی سلول های انسانی استفاده گردد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Subcloning and expression of SO9 gene, Saponaria Officinalis plant in E.coli and investigation of antibody titer in mouse

نویسندگان [English]

  • Amirhossein Barghi 1
  • Hossein Honari 1
  • Mohamadali Ebrahimi 2
  • Gholamreza Bakhshi Khaniki 3
  • Seyed Mojtaba aghaee 4
1 MSc in Plant Biotechnology, Science and Technology Center, Payam Noor University, Tehran, Iran
2 Associate Professor of Agricultural Sciences, Science and Technology Center, Payam Noor University, Tehran, Iran
3 Master of Systematic Vegetation, Science and Technology Center, Payam Noor University, Tehran, Iran
4 PhD. student of Bacteriology, Center for Science and Technology of Biology, Faculty of Basic Sciences, Imam Hossein Comprehensive University, Tehran, Iran
چکیده [English]

Introduction: Saponaria officinalis have various saponin isoforms. Saponin is a ribosome-inactivating protein (RIP). The SO9 isoform of saponins depurinates the adenine 4324 in the preserved GAGA sequence resulting in impairment of protein production. In this study, the S09 isoform was expressed in E. coli and its antibody titers were evaluated in Mouse.

Methods: The S09 gene was synthesized and isolated from the pUC57-S09 recombinant plasmid using the restriction enzymes BamH1 and Sal1, and then cloned in the expression vector pET28a(+). Expression of the new recombinant protein was induced by IPTG. The recombinant S09 protein was purified by Ni affinity chromatography. The recombinant protein was confirmed through western blotting. The Mouse were vaccinated through intraperitoneal injection of the purified protein and serum IgG titer was measured through ELISA.

Results: Subcloning of S09 gene in the pET28a(+) expression vector was confirmed by PCR and enzymatic digestion. The presence of 29 kDa protein band in SDS-PAGE showed the high expression of recombinant protein. The recombinant S09 protein was detected by polyclonal antibody. After injection of the protein to the test groups, the antibody titer was measured by ELISA.

Conclusion: The adjuvant property and immunogenicity of the purified recombinant S09 antigen showed that this antibody can be used to detect the presence of S09 in Saponaria officinal is, as a candidate for vaccine, for production of diagnostic kits, and in human cells anticancer studies.

کلیدواژه‌ها [English]

  • : Saponaria officinalis L
  • RIP
  • expression SO9
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دوره 27، شماره 1
فروردین و اردیبهشت 1399
صفحه 103-112
  • تاریخ دریافت: 20 اسفند 1397
  • تاریخ بازنگری: 16 اردیبهشت 1398
  • تاریخ پذیرش: 02 خرداد 1399
  • تاریخ اولین انتشار: 02 خرداد 1399