Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface
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. Accordi...
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| Format: | Article |
| Language: | English |
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Mazandaran University of Medical Sciences
2017-08-01
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| Series: | Journal of Mazandaran University of Medical Sciences |
| Subjects: | |
| Online Access: | http://jmums.mazums.ac.ir/article-1-9228-en.html |
| _version_ | 1797943700543766528 |
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| author | Shabanali Khodashenas Limoni Fatemeh Salimi Mehdi Forouzandeh Moghaddam |
| author_facet | Shabanali Khodashenas Limoni Fatemeh Salimi Mehdi Forouzandeh Moghaddam |
| author_sort | Shabanali Khodashenas Limoni |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T20:28:22Z |
| format | Article |
| id | doaj.art-355960d28cfd42389e0c45fbc9725ece |
| institution | Directory Open Access Journal |
| issn | 1735-9260 1735-9279 |
| language | English |
| last_indexed | 2024-04-10T20:28:22Z |
| publishDate | 2017-08-01 |
| publisher | Mazandaran University of Medical Sciences |
| record_format | Article |
| series | Journal of Mazandaran University of Medical Sciences |
| spelling | doaj.art-355960d28cfd42389e0c45fbc9725ece2023-01-25T07:50:58ZengMazandaran University of Medical SciencesJournal of Mazandaran University of Medical Sciences1735-92601735-92792017-08-01271511223Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome SurfaceShabanali Khodashenas Limoni0Fatemeh Salimi1Mehdi Forouzandeh Moghaddam2 Assistant Professor, Immunogenetic Research Center, Mazandaran University of Medical Sciences, Sari, Iran PhD Student in Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Associate Professor, Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 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.http://jmums.mazums.ac.ir/article-1-9228-en.htmlexosomelentiviral vectordarpinssoeing pcrlamp2b |
| spellingShingle | Shabanali Khodashenas Limoni Fatemeh Salimi Mehdi Forouzandeh Moghaddam Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface Journal of Mazandaran University of Medical Sciences exosome lentiviral vector darpins soeing pcr lamp2b |
| title | Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface |
| title_full | Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface |
| title_fullStr | Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface |
| title_full_unstemmed | Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface |
| title_short | Designing pLEX-LAMP-DARPin Lentiviral Vector for Exression of HER2 Targeted DARPin on Exosome Surface |
| title_sort | designing plex lamp darpin lentiviral vector for exression of her2 targeted darpin on exosome surface |
| topic | exosome lentiviral vector darpins soeing pcr lamp2b |
| url | http://jmums.mazums.ac.ir/article-1-9228-en.html |
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