| Ebola virus disease (EVD) is an acuteinfectiousdisease caused by the Ebola virus (EBOV). This disease is highly contagious and has a high morbidity and mortality,therefore, the Ebola virus belongs to high-risk pathogens and attracted international attention, it has been listed as a category A bioterrorism agent. Since EBOV was found, it had caused huge economic losses to the society. Since EVD were found, they had caused huge economic losses to mankind. There are no diagnostic reagents or vaccines which areapproved in thedomestic market at present. In addition, EBOV can be transmitted by aerosol actions and contacts, besides, the changes in the climate and ecological environments and the increase of international communication provide a convenient condition for its propagation. EBOV may spread to China at any time, therefore,it is very necessary to establish a rapid diagnostic method and develop high effective stockpile vaccines to prevent the outbreak of EVD.Part 1:Establish a rapid diagnosis and typing method of the EBOV. This study is aimed at the glycoprotein (GP) of the Zaire type and Sultan type Ebola virus to design consensus primers and probe, only one reaction to detect and discriminate the two lethal types of Ebola virus.Part 2:The establishment of neutralization test of Ebola pseudovirus. In order to effectively evaluate antibody neutralizing and avoid hidden danger on biosafety of EBOV as the pathogeny in the neutralization test, here, for the first time, VSVvirus packaging system and envelope proteins of Ebola virus were used to construct a pseudovirus. The identification result showed the pseudoviruses were consistent with EBOV reported in the literature on viral morphology and structure, and the result from inhibition assay of pseudovirus infection showed the EBOV pseudovirus infection was inhibited by EBOV antibodies. On this basis the neutralization test of EBOV pseudoviruses was established, and the test results indicated the neutralization test was able to effectively evaluate the antibody neutralization.Part 3:onstruction, identification and immunological experiment study of VLPs vaccine of EBOV, in recent years, virus like particles as a new vaccine have exhibited many unique advantages, indicating better development prospect. At present, the research of the EBOV VLPs is not yet being seen in the critical literature, therefore, the first recombinant baculoviruses (AcMNPV-GP-VP40) were constructed to express structural proteins (GP and VP40) of EBOV using Bac to Bac system by means of genetic recombination technology, the identification results showed the EBOV VLPs were expressed by the Sf9cells transfected with AcMNPV-GP-VP40. Immunological experiment results indicated that EBOV VLPs were able to induce the T cells activation and were able to cause the high levels of IL-2?IFN-??TNF-? and neutralizing antibody in mice; In addition, the EBOV VLPs were able to induce neutralizing antibody in monkeys, and has a high level of neutralizing effect. Therefore, these results showed that EBOV VLPs were able to stimulate strong cell-mediated immune response and humoral immune response in mice or monkeys.Part 4:Use the EBOV VLPs as antigens to immune horses with different adjuvants and hyper-immune serum were collected. Produce the purified horse anti-EBOV immunoglobulin under GMP conditions, the neutralizing antibody titer of immunoglobulin is above 1:20000.The purify of F(ab')2 is above 90%.In the end,the results of this studyshow that the Ebola virus fluorescence quantitative RT-PCR detectionmethod had excellent specificity andsensitivity;the EBOVVLPshas gooddevelopment potentialasvaccine; the purifiedhorse anti-EBOVimmunoglobulin we produce fit the demand ofthe nationalbiological products;the results of this studylaid the foundation for thecomprehensive prevention and control of EBOV. |
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