OBJECTIVE: To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope. METHODS: Single-ch...OBJECTIVE: To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope. METHODS: Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid.To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed. RESULTS: The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen. CONCLUSION: These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.展开更多
Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transf...Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection （TCUMGT） via ultrasound-targeted microbubble destruction （UTMD） to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles （MBs） with pcDNA3.l-human vascular endothelial growth factor 165 （pcDNA3.I-hVEGFls5） plasmids targeted to P-selectin （MB＋P＋VEGFp） were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups： microbubble only （MB）, microbubble＋P-selectin （MB＋P）, microbubble＋pcDNA3.l-hVEGF185 plasmid （MB＋VEGFp）, and microbubbie＋ P-selectin＋pcDNA3.1-hVEGF185 piasmid （MB＋P＋VEGFp）. The reverse transcription polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbent assay （ELISA） results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB＋P＋VEGFp delivery showed greater improvement than MB＋VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA3.1-hVEGF165 recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.展开更多
文摘OBJECTIVE: To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope. METHODS: Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid.To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed. RESULTS: The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen. CONCLUSION: These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.
基金Project supported by the Natural Science Foundation of Zhejiang Province(No.LY14H180003)the National Natural Science Foundation of China(No.81301231)the General Research Project of Zhejiang Provincial Department of Education(No.Y201636244),China
文摘Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection （TCUMGT） via ultrasound-targeted microbubble destruction （UTMD） to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles （MBs） with pcDNA3.l-human vascular endothelial growth factor 165 （pcDNA3.I-hVEGFls5） plasmids targeted to P-selectin （MB＋P＋VEGFp） were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups： microbubble only （MB）, microbubble＋P-selectin （MB＋P）, microbubble＋pcDNA3.l-hVEGF185 plasmid （MB＋VEGFp）, and microbubbie＋ P-selectin＋pcDNA3.1-hVEGF185 piasmid （MB＋P＋VEGFp）. The reverse transcription polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbent assay （ELISA） results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB＋P＋VEGFp delivery showed greater improvement than MB＋VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA3.1-hVEGF165 recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.
