Volume 27, Issue 151 (8-2017)
J Mazandaran Univ Med Sci 2017, 27(151): 12-23 |
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Abstract: (6687 Views)
Background and purpose: Exosome as drug delivery system is a novel and smart methodology enabling delivery of exosome cargo into specific tissue. This aim could be accessed by manipulation of exosome producer cells for expression of specific transmembrane-anchored ligand on exosomes surface. Accordingly, Lysosomal Associated Membrane Protein (LAMP) is one of the best choices for anchoring and chimerization with any ligand for this propose. In current study we designed a lentiviral vector which carries a chimeric gene for expression of LAMP2-DARPin in exosome to attach to HER2 on cancer cell surfaces.
Materials and methods: RNA was extracted from mouse skeletal muscle, then, cDNA was produced by RT-PCR and CDS of LAMP2b gene was amplified by specific primers. Two restriction sites were introduced between signal and mature peptide sequence by SOEing PCR. This fragment was inserted into pLEX-MCS lentiviral vector and cloned in E.coli. DARPin gene was designed, optimized and synthesized, then cloned between signal and mature peptide. Positive clone was confirmed by colony PCR and DNA sequencing.
Results: Electrophoresis of SOEing PCR product showed 1290 bp DNA fragment of LAMP2B CDS. Insertion of LAMP2 in pLEX vector was confirmed by electrophoresis and sequencing. Accordingly, DARPins was synthesized and inserted into pLEX-LAMP vector, electrophoresis and sequencing of purified plasmid from positive clone confirmed the insertion of DARPins into pLEX-LAMP vector.
Conclusion: We generated two lentiviral vectors, pLEX-LAMP for expression of any ligands in exosome surface and pLEX-LAMP DARPin for expression of DARPin on exosome surface for HER2 targeting.
Materials and methods: RNA was extracted from mouse skeletal muscle, then, cDNA was produced by RT-PCR and CDS of LAMP2b gene was amplified by specific primers. Two restriction sites were introduced between signal and mature peptide sequence by SOEing PCR. This fragment was inserted into pLEX-MCS lentiviral vector and cloned in E.coli. DARPin gene was designed, optimized and synthesized, then cloned between signal and mature peptide. Positive clone was confirmed by colony PCR and DNA sequencing.
Results: Electrophoresis of SOEing PCR product showed 1290 bp DNA fragment of LAMP2B CDS. Insertion of LAMP2 in pLEX vector was confirmed by electrophoresis and sequencing. Accordingly, DARPins was synthesized and inserted into pLEX-LAMP vector, electrophoresis and sequencing of purified plasmid from positive clone confirmed the insertion of DARPins into pLEX-LAMP vector.
Conclusion: We generated two lentiviral vectors, pLEX-LAMP for expression of any ligands in exosome surface and pLEX-LAMP DARPin for expression of DARPin on exosome surface for HER2 targeting.
Type of Study: Research(Original) |
Subject:
Biotechnology
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