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Research in Pharmaceutical Sciences، جلد ۱۰، شماره ۴، صفحات ۲۶۸-۰
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عنوان انگلیسی Expression of a biotin acceptor peptide-containing protein with potential incorporation on the lentiviral envelope as a viral surface engineering platform
چکیده انگلیسی مقاله Lentiviral vectors are among the promising viral based-vectors in gene therapy applications, but the efficiency of their targeting needs to be improved. (Strept)avidin-biotin adaptor system is a novel approach to modify the lentiviral envelope for better targeting properties. Herein, we describe utilization of this adaptor system by designing a candidate envelope protein-bearing biotin acceptor peptide (BAP) and evaluation of its expression in 293T cells. To this end, a DNA sequence containing flexible linkers, a 15-aminoacids BAP and specific membrane regions of a viral protein was designed and synthesized in tandem. The synthesized gene was amplified with polymerase chain reaction to include Bgl II and Sal I restriction sites and subcloned into the same sites of pDisplay vector in frame with HA-tag and myc epitope to construct the pDis-GS-BAP. 293T cells were transfected with pDis-GS-BAP and expression of resulting protein (dis-GS-BAP) was evaluated by Western blotting using anti-HA tag antibody. Efficiency of transfection procedure was evaluated by pEGFP-N1 vector and tracking for green fluorescent protein expression via fluorescence microscopy. Restriction analysis and DNA sequencing confirmed the precision of cloning steps. Fluorescence microscopy indicated above 70% transfection efficiency and Western blot analysis of pDis-GS-BAP-transfected 293T cells showed a protein band of approximately 17 kDa corresponding to the predicted size of dis-GS-BAP protein. These promising results indicate the possibility of cell surface expression and further biotinylation of dis-GS-BAP protein in ongoing studies.
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نشانی اینترنتی http://rps.mui.ac.ir/index.php/jrps/article/view/1634
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زبان مقاله منتشر شده en
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نوع مقاله منتشر شده Original Article
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