| The Postweaning Multisystemic Wasting Syndrome (PMWS) has been first reported at Canada in 1991, it has caused significant economic losses on swine industry around the world. Porcine circovirus type 2 (PCV2), one of the major pathogens of the PMWS is the smallest animal DNA circovirus. The disease is also related from various kind of virus, such as the Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Porcine Transfusion Transmitted Virus (PTTV), and Porcine Parvovirus (PPV) and so on. It is the effective way that we need to find out the distribution of viral genes and the trend to prevent the occurrence of disease. On the other hand, recently, the laboratory did a gene analysis about the PCV2 in China, and extracted the Consensus sequence, the sequence can improve the cross-protective function of vaccines. The experiment aim for improving the expression level, overcoming the disadvantages of not available for mass-produced due to the low titers of PCV2 when proliferated in cells. The consensus sequence is expressed by the baculovirus vector expression system that it needs to proliferate in insect cells, we target to increase the expression level by improving the condition of cell culturing.The study includes three parts, the samples for testing mainly come from the pig herds which have clinical symptoms. Starting from the epidemiological point of view, the multi-pathogen of PMWS are detected, besides, more precisely to describe a distribution of PCV2 genotype of the targeted areas. The last part of the lab is to enhance the expression of recombinant baculovirus by improving the conditions of insect cell culturing. The experiments as follow:1. Epidemiological investigation of the post-weaning multisystemic wasting syndromeA total number of 970 tissue samples are collected from large farms in Xuzhou, Qidong and Taizhou. Using the PCR method, the investigation detects PMWS associated pathogen. The result shows that all samples, positive rate of PRRSV is 20.88%(90/431), positive rate of PCV2 is 25.36%(246/970), positive rate of PTTV1 is 42.65%(316/741), positive rate of PTTV2 is 29.55%(219/741), and positive rate of PPV is 3.5%(11/316). Positive rate of PCV2 and other pathogens co-infection is between 19.8% and 22.9%, TTV1 and TTV2 co-infection rate reaches as high as 31.07%, overall the PCV2 prevalent in these areas.2. Molecular epidemiology investigation of the porcine circovirus type 2There are 24 samples which are all positive for PCV2 extended the full length by using PCR, the full-length sequence determination analysis reveals that the 24 samples itself related to the PCV2 genome sequence homological rate is between 95.1% and 100%, and compare with the strains PCV2a(AF055392) alignment, the rate of homology between 95.1% and 96.7%, compare with the strains PCV2b(AF055394) alignment, the rate of homology between 95.2% and 99.4%, compare with the strains PCV2c(EU148503) alignment, the rate of homology between 93.8% and 95.1%.3. Improving Sf9, High 5 cell culture conditions to enhance the expression of the recombinant baculovirus vFBHYHO1-2/vFBHYHO2"With rapidly developing, insect cell culture as a biochemical engineering skills has been more important in eukaryotic baculovirus expression system. How to deal with the corresponding recombinant baculovirus, select the appropriate culture conditions that can apply to the commercialization are the important research directions in recent years. The experiment tries to improve the conditions of culturing the Sf9 and High5 insect cells, meanwhile, in order to improve the viral protein expression, adjust some factors to enhance the expression of the recombinant baculovrius vFBHYHO1-2/vFBHYHO2... |
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