Gene Transfer Of The Recombinant Plasmid PEGFP-vp5 Using Carbon Nanotubes As Gene-transfer Vectors

With the in-depth study of gene function, bacterial transformation, transfection of cells,etc. has become the basic method of gene function research in lab work. Looking for a carrying rate, wear strong membrane capacity, low cost, small-cell-toxic carrier as gene transfer vector is of great significance for gene function studies and applications. The functionalized carbon nanotubes, such as ammonium or carboxyl-functionalized carbon nanotubes, can provides multiple sites for the attachment of drugs, amino acids, proteins and other substances, and brought them into the cells to play a biological role. The vp5 gene express capsid protein VP5 of grass carp reovirus(GCRV), the immunogenic vp5 gene as a DNA vaccine has been shown in previous studies. In this work, we constructed the CNTs-NH3+(MWCNTs-NH3+and SWCNTs-NH3+):plasmid pEGFP-vp5 complexes by special chemical modification method. The complexes were applied as a material of E. coli(BL21)transformed, grass carp kidney cells(CIK) Transfection and grass carp juveniles immunoassays to screen for gene carrier system and verify its gene transfer effect by transfection efficiency, immune protection et al. The results obtained are as follows:1. The vp5 eukaryotic expression system(pEGFP-vp5) was constructed from outer membrane protein gene(vp5) of GCRV as the template by molecular biology techniques. By the study of four MWCNTs-NH3+(S1、S2、S3、S4): plasmid pEGFP-vp5 complexes to transform E.coli(BL21), we found that the S4 functionlized with a pyrrolidine ring bearing a free amino-terminal oligoethylene glycol or N-hexyl moiety attached to the nitrogen atom has an excellent conversion performance. The optimum ratio(S4:DNA) is 8:1; appropriate amount of recombinant plasmid pEGFP-vp5 is about 20 ng; and conversion is simple and low cost. It illustrated that amino-MWCNTs are expected to become an excellent bacterial transformation vector.2. By the study of four MWCNTs-NH3+(S1、S2、S3、S4): plasmid pEGFP-vp5 complexes to transfect grass carp kidney cells(CIK), Agarose gel shift assay results show that ammonium group-functionalized carbon nanotubes are able to condense pEGFP-vp5 in varying degrees. Scanning electron microscope images shows that CIK cells show a great affinity for MWCNTs-NH3+and the CNTs covering the cell surface tend to orient their tips perpendicularly to the cell surface, and appear to be “needle-pricking the cells”. Transmissionelectron microscope images confirmed that MWCNTs-NH3+penetrate the cell membranes and are widely dispersed in the CIK cell. Subsequent studies confirmed that both functional groups and charge ratio are important factors that determine the transfection efficiency of plasmid DNA. We found that the S4 has an excellent cell transformation performance. The optimum ratio(S4:DNA) is 6:1 All these results indicated that amino MWCNTs are expected to become an excellent cell transformation vector.3. Using the chemical modification methods, the pEGFP-vp5 were link to the surface of CNTs(S4 and SWCNTs-NH3+) to prepare CNTs-pEGFP-vp5 DNA vaccine system.Intramuscular and bath immunization for grass carp fingerlings showed: S4 is not suitable as a pEGFP-vp5 vaccine vector; for SWCNTs-NH3+, higher levels of transcription and expression of vp5 gene could be detected in muscle tissues of grass carp in SWCNTs-pEGFP-vp5 treatment groups compare with naked pEGFP-vp5 treatment groups. Moreover, antibody levels, immune-related genes, and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pEGFP-vp5 vaccine. In addition, we found that a good immune protective effect was observed in bath immunization group; which at a concentration of 20 mg/L could reach the similar relative percentage survival(approximately 100%) in injection group at a dose of 5 μg. SWCNTs-pEGFP-vp5 bath treatment group can also reach the similar immune protection in injection treatment group. All these results indicated that ammonium-functionalized SWCNTs could be used as an effective vehicle for aquaculture DNA vaccine and it provide extensive application prospect to aquatic vaccine and might be used to vaccinate fish by intramuscular injection or bath administration method.In summary, for the cellular permeability of carbon nanotubes, amino-CNTs functionlized by aspecial chemical modification methods can be used as carrier to transform E.coli(BL21), transfect grass carp kidney cells(CIK), immune grass carp juveniles. The application of CNTs-NH3+:DNA complexes was laid the foundation by this work.