| Grass carp reovirus(GCRV), the causative pathogen of grass carp hemorrhagic disease, can cause fatal hemorrhagic disease in yearling grass carp(Ctenopharyngodon idella). To prevent the occurrence of virus disease, lots of measures have been adopted, such as the production of specific pathogen free(SPF) and the use of vaccines. However, the high production costs and long production cycle restricted the development of the SPF technology. Injection immunization needs lots of human, material, time and financial resources, which limits its application. Nano drug delivery systems have high drug loading capacity to penetrate tissue barriers to achieve efficient transport of drugs. In this study, single-walled carbon nanotubes(SWCNTs) as carriers were used to manufacture SWCNTs drug delivery systems, SWCNTs subunit vaccine and DNA vaccine delivery systems by chemical modification method. Then, these delivery systems were used to evaluate their effects on the prevention of grass carp hemorrhage. Meanwhile, we explored the mechanism of SWCNTs for transport across fish cell, and the developmental toxicity of SWCNTs on fish was also examined. The results obtained in this work were as follows: 1. Study of SPF for grass carpUsing the chemical modification methods, the antiviral drugs(ribavirin and moroxydine) were link to the surface of SWCNTs to prepare R-SWCNTs and M-SWCNTs. High performance liquid chromatography(HPLC) analysis showed the drug carrying capacity of R-SWCNTs and M-SWCNTs were 20.45 ± 0.36% and 32.41 ± 0.24%. Medicated bath treatment showed that: when antiviral drugs concentrations arrived at 10 mg/L in R-SWCNTs and M-SWCNTs treatment groups, the survival rates were more than 90% and the infection rates dropped to 0 for the fry and fingerling of grass carp. However, even the naked antiviral drugs(ribavirin and moroxydine) concentration reached to 20 mg/L, survival rates were less than 35% and infection rates were about 50% for grass carp. Drug metabolism studies showed that: compared with naked antiviral drugs treatment groups, the drug content increased about five times and metabolic time prolonged about six times in larvae, the drug content increased about three times and metabolic time prolonged about two times in fish in R-SWCNTs and M-SWCNTs treatment groups. 2. SWCNTs as candidate recombinant subunit vaccine carrier for immunization of grass carp against grass carp hemorrhagic diseaseUse outer membrane protein(VP7) of GCRV as the template to build VP7 prokaryotic expression system by molecular biology techniques, and prepare VP7 protein(47.8 KDa) by fermentation and purification technology. Using the chemical modification methods, the VP7 protein were link to the surface of SWCNTs to prepare SWCNTs-VP7 subunit vaccine system. Quantitative protein analysis showed that the protein content of VP7 in SWCNTs-VP7 was 48.9%. Different developmental stages of grass carp(0.2 and 25 g) injection and bath immunization results showed that: for the same doses or concentrations of vaccines, the antibody titers were significantly improved(P<0.05), antibody retention times were significantly extended(P<0.05), immune-related physiological indicators(respiratory burst, lysozyme and complement) activities were significantly increased(P <0.05), immune related genes expression(Ig M, Ig D and MHC-I, etc.) were significantly increased(P<0.05). After challenged with GCRV at 21 d post immunization, the immune protection rates reached to 90% in SWCNTs-VP7 bath(20 mg/L) and injection(1.0 μg for 0.2 g grass carp, 100 μg for 25 g grass carp) immunization groups for two kinds of grass carps. However, the immune protection rates were less than 25% for 20 mg/L VP7 bath immunization groups, less than 40% for VP7 injection(1.0 μg for 0.2 g grass carp, 100 μg for 25 g grass carp) immunization groups. Thus, SWCNTs can improve the protection rate of subunit vaccine, and SWCNTs-VP7 bath immunoassay group can also reach the similar immune protection in injection immunoassay group. 3. SWCNTs as candidate DNA vaccine carrier for immunization of grass carp against grass carp hemorrhagic diseaseUse outer membrane protein gene(vp7) of GCRV as the template to build vp7 eukaryotic expression system(pc DNA-vp7) by molecular biology techniques. Using the chemical modification methods, the pc DNA-vp7 were link to the surface of SWCNTs to prepare SWCNTs-pc DNA-vp7 DNA vaccine system. Intramuscular and bath immunization for grass carp fingerlings(25 g) showed: under the same immunizing dose or concentration, antibody titers were significantly increased in fish(P<0.05), immune-related physiological indicators(respiratory burst, complement activity and superoxide dismutase) activities were significantly increased(P<0.05), the expression of immune-related genes(Ig M, CD8α and MHC-IIB, etc.) were significantly increased(P<0.05) in SWCNTs-pc DNA-vp7 DNA vaccine system. After challenged with GCRV at 28 d post immunization, the immune protection rates reached to 100% in SWCNTs-pc DNA-vp7 bath(10 mg/L) and injection(10 μg for each grass carp) immunization groups for grass carps. However, the immune protection rates were less than 30% for pc DNA-vp7 immunization groups. Thus, SWCNTs can reduce the useage of DNA vaccines, and SWCNTs-pc DNA-vp7 bath immunoassay group can also reach the similar immune protection in injection immunoassay group. 4. Transmembrane and metabolism mechanism of SWCNTsUsing carp epithelial tumor cell line(EPC) as the cell model, fluorescein isothiocyanate- labeled SWCNTs(F-SWCNTs) as the material, the transmembrane mechanism of SWCNTs were measured by flow cytometry and fluorescence imaging techniques. The results showed that: the transmembrane mechanism of SWCNTs included energy consumption and direct cell membrane penetration. For energy consumption, clathrin-mediated endocytosis and macropinocytosis play the important roles, while the envelope-mediated endocytosis had little effect. Fluorescence and Raman spectroscopy study showed that: after EPC cells treated with F-SWCNT(100 mg/L) for 2 h, the content of SWCNTs can reach to 24.71×10-6 pg/cell. After removing F-SWCNT, the content of SWCNTs decreased gradually, was 0.47×10-6 pg/cell at 48 h. Meanwhile, it also found the ratio of D and G peaks(Ramean spectra) for SWCNTs were increased from 0.18 to 0.91, and lots of SWCNTs observed in lysosomal. These results indicated that SWCNTs can metabolism and degradation in EPC cell, and the degradation mechanism related to lysosomal structure. 5. Safety evaluation of SWCNTs on fishThe developmental toxicity of SWCNTs on rare minnow showed that: SWCNTs had little toxicity on fish embryo, the 144 h LC50 and EC50 values on larvae were 140.8(109.3-174.2) and 109.8(85.1-135.1) mg/L. When SWCNTs concentration arrived at 100 mg/L, the body length and heart rate were significantly reduced, swimming speed were increased(P<0.05), glutathione content and catalase activity were significantly reduced(P<0.05), malondialdehyde content, superoxide dismutase glutathione S-transferase, reactive oxygen species, DNA damage, Caspase-3, 8 and 9 activity significantly increased(P<0.05), the expression of hsp70, apaf-1, bcl-2, caspase-3 and caspase-9 were significantly increased(P<0.05). The results of F-SWCNTs metabolism in larvae showed that: F-SWCNTs can be gradually enriched in the larval body, but after removal of F-SWCNTs, F-SWCNTs can be completely removed from the larvae.In summary, SWCNTs as a nano carrier can effectively carry antiviral drugs to against GCRV in grass carp; can effectively carry genetically engineered vaccines to immune grass carp by bath and injection immunization. Meanwhile, SWCNTs have little toxicity on fish, and have broad applications for the production of SPF and genetically engineered vaccines. |
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