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" Construction, cloning, expression, and purification of DT386-BR2 fusion protein in Escherichia coli and evaluation of its biological activity "
Fatemeh Shafiee
Mohammad Rabbani, Ali Jahanian-Najafabadi
Mahdi Behdani
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Isfahan University of Medical Science
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Latin Dissertation
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English
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| Record Number
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111259
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T17841
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QU55,S525c,2016
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| Main Entry
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Shafiee , , Fatemeh
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| Title & Author
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Construction, cloning, expression, and purification of DT386-BR2 fusion protein in Escherichia coli and evaluation of its biological activity\ Fatemeh Shafiee
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| College
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school of pharmacy and pharmaceutical
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2016
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Ph.D
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| field of study
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Pharmaceutical Biotechnology
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137p.
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Research Project No: 193038
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فاطمه شفیعی
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| Abstract
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BR2 is a buforin derivate antimicrobial peptide with the ability to act as a cell penetrating peptide. So this peptide can be used as a carrier for delivering therapeutic molecules to cells. The aim of this study was to produce a fusion protein consisting of the catalytic and translocation domain of diphtheria toxin as the toxic moiety of immunotoxin, fused to BR2, as the targeting moiety for targeted drug delivery to different cancer cell lines.Methods:Fusion protein design was performed using Modeller 9.14 and the cloning of DT386-BR2 and DT386 genes to pET28a vector achieved by NcoI and XhoI restriction enzymes. Expression of these recombinant proteins was induced using IPTG (1 mM), and optimization of the expression was performed based on the selection of the best host, cultivation condition and culture medium. Purification of the recombinant proteins was performed using nickel affinity chromatography. The correct bands were confirmed using Western blot analysis and finally, MTT assay was used in order to evaluate the cytotoxicity of DT386-BR2 fusion protein on different cell lines.Results:The best predicted model for the target fusion protein using multiple template modeling (Modeller program) had a minimum DOPE score (-43479.42969) and molpdf (1877.55469). Digestion of the recombinant pET28a containing inserts using NcoI and XhoI restriction enzymes created a band approximately in 1200 bps for DT386 and 1290 bps for DT386-BR2 respectively. Analyzing the purified recombinant protein on 12% SDS-PAGE showed a band of approximately 45 kDa for DT386 and 47 kDa for DT386-BR2, respectively which confirmed by Western blot analysis. Finally, MTT assay results showed that this fusion protein had concentration dependent effects on cancer cell line and its effect on normal cells (HUVEC) was significantly lower than cancer cells. Conclusion:We could successfully design and produce a fusion protein as immunotoxin with the ability of specific toxicity to cancer cells without significant cytotoxic effects on normal cells. Thus this fusion protein may be a potential candidate cancer targeted therapy.
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| Descriptor
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fusion protein
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Escherichia coli
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Diphtheria toxin
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Buforin
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فیوژن پروتئینی
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توکسین دیفتری
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اشرشیاکولی
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بوفورین
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Rabbani , Mohammad
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Jahanian-Najafabadi , Ali
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Behdani , Mahdi
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Isfahan University of Medical Sciences
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| Translated Title Supplied by Cataloguer
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طراحی، بیان و خالص¬سازی فیوژن پروتئین DT386-BR2 در Escherichia coli و ارزیابی اثرات بیولوژیک آن
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