| PRRS(Porcine Reproductive And Respiratory Syndrome) is an infectious disease with high infection rate, seriously affecting the pig industry in the world. PRRSV is divided into the European type PRRSV(genotype 1) and America type PRRSV(genotype 2), the difference of gene homology between the two genotypes is about 40%. The main clinical symptoms of PRRS are reproductive disorder in sows and prevalence of pneumonia, growth retardation and mortality increased in weaning piglets. Many countries have been using the attenuated vaccine and inactivated vaccine to prevent PRRSV. The risk not only attenuated vaccine has possibility of virus, and also the immunogenicity of inactivated vaccine is generally difficult to effectively prevent PRRSV. Therefore, we need to develop a safe and effective PRRS vaccine aiming at PRRSV with the characteristics of genetic diversity. The main epidemic strain of PRRSV in China is American type PRRSV, but in recent years, European type PRRSV has been isolated in many provinces. The emergence of European type PRRSV should attract our attention. All of the PRRS vaccines in our country are American type which produced poor cross immune protection in the European type PRRSV. So we need to develop a safe and effective PRRS vaccine in European type PRRSV.Porcine circovirustype 2(PCV2) is the main pathogen of Post-weaning multisystemic wasting syndrome(PMWS), which is divided into PCV-1 and PCV-2. PMWS is recognized as one of the most important infectious diseases of swine industry in the worldwide from its first report in Canada in 1991, and now it has spread to many countries in the world. Therefore, researching and developing a safe and effective vaccine is the key to solve the problem of PCV2 epidemic. Objective:This study developed researches of preparation and immunological of recombinant vaccinia virus vaccines against European-PRRSV and PCV2. Prepare European type PRRSV and PCV2 recombinant vaccinia virus vaccine structured in laboratory by using micromirrier bioreactor technology, and evaluate the immune effect by pig body immunity test. Contents:1. Screen seed cells and virus with strong proliferation ability to establish cell bank and virus library.2. Optimize preparation conditions of recombinant vaccinia virus vaccine: cell culture conditions, infection conditions and production parameters.3. Establish three production processes, including batch culture, Semi-batch culture and perfusion culture, to prepare recombinant vaccinia virus vaccine against European-PRRSV and PCV2 by bioreactor with microcarrier technology.4. Evaluate immune efficiency of pig body immune recombinant vaccinia virus vaccine against European-PRRSV and PCV2 prepared by microcarrier bioreactor system. Methods:1. Establishment of cell and virus bankScreen out safe, non-tumorigenic BHK-21 cell and capable of producing European-PRRSV and PCV2 recombinant vaccinia virus r VTT-ORF2-EUO3O5 strain, via cell passage, cell infection, and virus titration method, etc. Establish seed cell bank and virus library to lay a good foundation for further optimization of production process.2. Optimization preparation conditions of recombinant vaccinia virus.Identify a set of production control conditions that can be used for cell proliferation and viral amplification through the optimization of cell conditions and infection conditions. Future optimize control parameters by studying the effects of temperature, p H, rotation speed and dissolved oxygen on cells and virus yield in the course of training to provide effective parameters for large-scale cell culture and virus proligeration.3. Preparation of recombinant vaccinia virus vaccineMake process of bioreactor production through combining the data of recombinant vaccinia virus vaccine after optimization. Select the best production process by the effect of glucose consumption and lactate production on cell proliferation in the production process. Test the vaccines to determine whether the quality is consistent with the third Department of Veterinary Medicine. Provide reference for the preparation of viccinia virus vaccine.4. Study on immunity of recombinant vaccinia virus vaccine in swine.Recombinant vaccinia virus(r VTT-ORF2-EU-O3O5)based on ORD3 and ORF5 genes of classical strain LV of European type PRRSV and ORF2 gene of PCV2 carried on immune experiment. Detect the immune index to analyze recombinant vaccinia virus vaccine. Results:1. The safety and purity of BHK-21 cells were proved by the tumor experiment. Comparing the production of seed cells and seed virus production verifies the dominant of cell and virus proliferation. It is proved that European type PRRSV and PCV2 recombinant vaccinia virus could express the exogenous gene and had no difference with natural vaccinia virus in appearance by PCR and WB. The seed cell bank and seed bank were successfully established, and 16 strains seed cells and 10 strains seed virus were frozen.2. Through the cell conditions optimization, finally DMEM medium with 10% serum(Hyclone) was determined to culture cells. Through the viral infection conditions optimization, the dose of infection of 0.5MOI and 2% serum concentration DMEM was used as a viral infection conditions. Through the optimization of production parameters, the infection stage parameters were determined to be 37 degrees Celsius, p H=7.3 and M=80r/min at the stage of cell proliferation, and 35 degrees, p H=7.1 and M=40r/min at infection stage.3. Establishing three kinds of production methods through using microcarrier and bioreactor apparatus, including batch culture, Semi-batch culture and perfusion culture. According to the effects of nutrients and metabolism on cell proliferation, determine the perfusion culture is the most suitable for the preparation of vaccine production. The quality of recombinant vaccinia virus vaccine prepared by bioreactor was in line with the third Department of Veterinary Medicine.4. Through the pig body immunity, the European type PRRSV and PCV2 two recombinant pox virus vaccine immunization group could induce specific immune responses in pig in 35 days with the neutralizing antibody titer of 1:18, showing that the recombinant vaccinia vaccine can induce humoral immune responses. The results of cell factor and T lymphocyte proliferation test showed that the level of immune group was significantly higher than that of wild poisonous and PBS control group, indicating that the recombinant vaccinia virus vaccine can induce the immune response of the organism. In the challenge experiment, the body temperature of the vaccine immunization group was significantly lower than that of the control group, and the temperature was lower than 40℃. The viral load and the pathological results showed that the recombinant vaccinia virus vaccine can provide good protection... |
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