Construction, Characterization And Application Of A Novel Nano-gene Vcctor And Protein Detection Nanoprobes

Nanomaterials have been broadly studied because of their unique properties such as small size,large specific surface area and high surface activity. By combining nanomaterials with biotechnology, itprovides a new prospect for studying life science and solving biological and medical problems with newapproaches and methods. In the study of gene delivery, the application of conventional gene vectors islimited by their inherent disadvantages including receptor-dependent host tropism, immunogenicity, andlow efficiency and loading capacity. Magnetic nanoparticles, with the features of low toxicity, non-immunogenicity, and high DNA loading and protecting capacity, can be used as novel gene deliveryvectors for animal somatic cells to overcome deficiencies of conventional gene vectors. On the aspect ofprotein detection, antibody-based protein detection methods are limited by the interspecies cross-reactivity and the availability of antibodies from different species. DNA nanobarcodes, as a novel DNAnanomaterial which can be easily labeled, modified and manipulated, can be used to fluorescently labelantibodies to surmount the cross-reactivity and low availability problems for multiplexed proteindetection. Based on the above mentioned two points, we respectively chose PEI coated magneticnanoparticles to construct gene vectors for animal somatic cell gene delivery and DNA nanobarcodes todevelop novel protein detection nanoprobes for multiplexed protein detection. The major contents aresummarized as follows:1. The characteristic of PEI coated magnetic nanoparticles (NPmag) were characterized by SEM,AFM and laser particle size analyzer. The results showed that NPmagwere uniform spherical particleswith good dispersion and an average diameter approximate150nm. NPmagwere positive charged with azeta potential of+29.4mV, which enabled them to electrostatically bind with DNA. The diameter of theparticles increased to approximate200nm, zeta potential drop down to+23.1mV after NPmagand DNAformed complexes. The DNA binding and protecting abilities of NPmagwere investigated via gelretardation assay. The results demonstrated that NPmaghad a high DNA binding capacity and nucleasecleavage protecting capability. The cytotoxicity of NPmag/DNA complexes to PK-15cells was tested byMTT assay. NPmag/DNA complexes showed no obvious negative effect on the cell viability of PK-15cells. The above characterization and investigation results showed that PEI coated magneticnanoparticle, NPmag, would be an excellent nano-gene vector for gene delivery.2. To evaluate the performance of NPmagas gene delivery vector for transforming animal somaticcells, we used PK-15cell as a model cell and delivered GFP gene to PK-15cells using NPmagas genevector. The results showed that the GFP transfection efficiency was dramatically enhanced byemploying magnetic field during the process of delivery. The optimal transfection condition for PK-15was achieved by preparing NPmag/DNA complexes for transfection at1:1mass ratio. G418was utilizedto screen the neomycin-resistant cell clones to obtain transformed cell lines expressing GFP proteins.PCR result showed that the exogenous GFP gene was integrated into the genome of PK-15cells. Theachievements of using NPmagas gene delivery vector to transform PK-15cells provided an important experimental basis for target gene delivery to porcine embryonic fibroblast and using somatic cellnuclear transfer technique to breed transgenic animals. By using NPmagas gene vector, pEGFP-N1andDsRed plasmids were co-delivered into PK-15cells, the transfected cells can express green fluorescentprotein (GFP) and red fluorescent protein (DsRed) simultaneously, which laid the foundation for thestudy of large fragment gene and multiple gene delivery in the future.3. A novel DNA nanomaterial, DNA nanobarcode, was obtained by hybridizing fluorescent labeledoligonucleotides. DNA nanobarcodes have signal coding capacity based on the color ratios of differentfluorescent labels. EZZ protein which can bind to most IgG antibodies was conjugated to anoligonucleotide linker to form DNA-EZZ hybrid molecule. Using DNA-EZZ as a universal adapter,novel protein detection nanoprobe, IgG nanobarcode, was constructed by connecting DNA nanobarcodeand IgG antibody together. The antigen binding capacity of IgG nanobarcodes was investigated via IgGantibody competitive assay. The result showed that the antigen binding capacity of IgG nanobarcodeswas same as the unlabeled IgG antibodies.4. Various protein detection methods, such as dot blotting, micro-beads based protein detection andimmunohistochemical assay were conducted by utilizing IgG nanobarcodes as detection probes. Thesuccesses of single and multiplexed proteins detection in these methods provided an experimental andtheoretical basis for the practical application of IgG nanobarcodes in the future. We further developedQD labeled and HRP labeled probes using DNA-EZZ as adapter. Western blot were employed to test thefunction of these probes. It was shown that the QD or HRP labeled probes were fully functional andcould be used to realize protein detection with diverse read-out methods.