| Subgroup J of avian leukosis is a disease caused by the J subgroup avian leukemia virus(ALV-J), which is belong to the retrovirus. Since 1988, ALV-J has been widely spread in chicken flocks in the world. Especially since 2009, ALV-J has been in some layers in our country, the incidence rate is as high as 60%, the mortality rate is over 30%, which is very harmful to poultry industry. Because of the relatively close genetic characteristics of different strains, different genetic characteristics of the virus caused by different tumors, so far there is no effective method to prevent and control ALV-J. because of the vertical transmission of ALV-J, the immune tolerance of the innate infection is the main source of infection, there is no vaccine. The effective measures to control J subgroup avian leukemia are the elimination of the main RNAi, and the small molecule nucleic acid drugs have broad prospects in the prevention and control of the vaccine. ALV-J in the env gene of NX0101 gp85 strain siRNA, the siRNA gene was cloned into the miRNA framework. The p-miR-env gene was cloned into pc DNATM6.2-GW/Em GFP-miR eukaryotic expression vector. The results confirmed that the RNAi gene therapy was effective, but thein vivo efficiency of RNAi was a great challenge.So far, the RNAi system has a viral vector system and a non viral vector system. Although the virus vector has high transfection efficiency and long time in vivo. But it has a very serious security problem, and the purpose of the gene is small, and the preparation is complex. The cost is high, and it can't be used repeatedly, so it is limited in clinical application. The non viral gene delivery system, such as nanometer particles, in the same time, can be modified to increase the time of body function, so as to avoid the clinical safety barrier, and accelerate the clinical treatment of siRNA drugs. Chitosan as the only naturally occurring positively charged polymer chitosan, it also has low cell toxicity, no immunogenicity, etc., in the presence of the enzyme in vivo is stable, protection combined with gene, and according to the needs of the process of degradation, and degradationproducts without side effects. In addition, because the diameter of the animal cell is about the size of the nucleus is usually smaller than that of the large volume of foreign gene vector complex cells, the ideal compound volume should be controlled at the nanometer level. According to the molecular weight of chitosan, the degree of chitosan and the amount of chitosan and gene, the size of the chitosan gene complex can be controlled to a few hundred nanometers, and thus the animal cell is smooth. Besides, under physiological conditions, other non viral loads and serum proteins, which lead to the efficiency of gene transfer efficiency, and chitosan in the serum or body fluid environment has kept a very high gene transduction efficiency. Therefore, the application of chitosan in gene delivery system is highly praised.In view of the good biocompatibility and biodegradation of chitosan, we choose the pmiR-env recombinant plasmid targeting ALV-J gene, which can effectively protect ALV-J from siRNA cells and can effectively protect the CEF cells and provide a new exploration for the treatment of avian leukemia virus. It can effectively protect the virus.Using our laboratory preparation of P-miR-env recombinant plasmid transformation, to large endotoxin, adopting compound condensing prepared CTS-P-miR-env nanoparticles.By transmission electron microscopy(sem) negative dyeing observation nanoparticles morphology and size;Particle size analysis meter measuring nanoparticles size;Trace ultraviolet spectrophotometer determination of CTS-p-miR-env supernatant of the content of free plasmid calculate coating rate;Gel electrophoresis method for determining the stability of nanoparticles;Gel electrophoresis method to analyze the stability of nanoparticles in fetal bovine serum.Through the experimental study in CTS-p DNA nanoparticles in ALV-J virus infection chicken body influences the effects of viral replication, use the preparation of good CTS-p-miR-env nano preparation in animal experiments, SPF chicks were divided into 8 groups, one group as the blank control group;Two groups to take poison control group;Three groups of negative control group;Four groups of CTS-p-miR-env nano preparation group;5 groups of naked plasmid control group;6 for CTS-p-miR-env nano preparation low dose group;7 for CTS-p-miR- dose group in env nano preparation;8 for CTS-p- miR- env nano preparation high dose group;Picking cotton swabs detect ALV-J p27, to detoxify the condition monitoring;Through the comparison and hematology detection, gross weight to weigh disease assessment such as observation and histological observation CTS-p-miR-env the antiviral effect of nano preparation in the body.CTS-P-miR-env nanoparticle preparation results:(1) the successful preparation of the CTS-P-miR-env nanoparticles, the electron microscope found that nanoparticles are spherical, shape is consistent;(2) particle size analysis measuring instrument to determine the particle size of 90% for 102 nm, the average particle size is 88 nm;(3) coating at a rate of 86%;(4) electrophoresis results showed that the CTS-p-miR-env nanoparticles stable properties, can prevent fetal bovine serum Rnase degradation of siRNA, protective effect of siRNA. Thus ion gel method to the preparation of uniform particle size distribution, shape rules, has a higher rate of coating CTS-p-miR-env nanoparticles.CTS-p-miR-env protection, nanoparticles of siRNA with enzyme solution for gene delivery vectors.The results of vivo experiment:(1) p27 experimental results showed the group of inoculation were antigen-positive, the control group were negative, naked DNA inoculation group showed the arise of detoxification is in 20 days and 24 days, Nano formulation dose inoculation groups appeared in 28 days detox, high-dose formulation of nano-inoculation group does not appeared detoxification, S / P value had been low;(2) in the present experiment, the number of white blood cell of the ALV-J inoculation group reduced and indicated that the virus made harmful effects on the bone marrow hematopoietic system leading the declining of the function of the chicken hematopoietic, thereby reducing the total number of white blood cells, the decreasing trendof white blood cell and the decreasing trend of the lymphocyte ratio is similar. the number of white blood cell of naked DNA inoculation control group and nano formulations inoculation control group was significantly higher than inoculation group, indicating the protective effect on bone marrow; We also found that nanoformulation inoculation group consisting of high dose to low dose followed by the number of leukocytes increased, and were higher than naked DNA inoculation group, indicating the protective effect of nano formulations of the body is stronger than naked plasmid;(3) lt is can be seen that the heart, spleen, liver, thymus, fabricius of inoculation group atrophy significantly trough gross necropsy lesions, the heart, spleen, liver, thymu s, bursa of nanopreparation inoculation group from low doses to high doses of gradually become normal, but better compared to naked plasmid group;(4) it is can be seen that myocardial of inoculation bleeds obviously,lymphocyte inflammatory forms, a large number of lymphocytes in liver inflammatory forms; renal hemorrhage; lung congestion, interstitial widened; the spleen lymphocytes loss, the number reduced significantly. The tissue of the blank control group was normal. It showed minimal change in naked plasmid group and low dose inoculation group of nanotechnology formulation, nano-dose and high-dose formulation inoculation with normal control group were similar.From that, this experiment was successfully completed. Morphology showed a higher rate of coating. siRNA has a protective enzyme solution. Fit for as a gene delively vector. Through the experiment in vivo p-mir-env, recombinant expression plasmid has the inhibitory effort of leukemia virus replication. Nano preparation and inhibitory replication. Nano preparation and inhibitory effort of leukemia virus replication is better than that naked plasmid. The research solved siRNA the problem such as poor stability, particle size small, not easy to enter the target cells, and other virus for it provides a new effect prevention and treatment. |
PDF Full Text Request