| Poliomyelitis is an acute infectious disease caused by the polio virus infection.The motor neuron damage of the patients especially pediatric patients may appear limbs flaccid paralysis due to the virus invasion of nervous system.And currently there is no effective drug treatment of polio virus infection,mainly preventable by immunization vaccines.There are only three countries being in the risk of polio virus infection now,since the using of Salk inactivated vaccines and Sabin attenuated live vaccines from the 1950 s.The Global polio eradication initiative(GPEI)plans to achieve the goal of eradication of poliovirus by the end of 2018,and the polio virus will be gradually phased out.So a new method of studying polio virus and evaluating the efficacy of polio vaccines should be established after the virus sequestration.In this study,we packaged the pseudovirus of three serotypes of wild poliovirus(Mahoney,MEF-1,Saukett)and Sabin2 strain,which can replace the original virus for virological research and vaccine efficacy evaluation test of poliovirus.In this study,the pseudoviruses of three serotypes of wild polio virus and Sabin2 strain were packaged by the replicon-RNA and capsid-plasmid co-transfection in 293 FT cell.Firstly,construct green fluorescent protein EGFP and capsid protein P1 infusion expression vector.Then replace the P1 gene with the luciferase gene to obtain the defective replicon vector and converted into RNA form by the in vitro transcription kit before transfection of cells.Secondly,the capsid expression plasmid and the replicon-RNA were transfected into 293 FT cells sequentially.24 hours lately,the pseudovirus can be collected by repeated freezing and thawing cells.Finally,we identified the pseudovirus by the experiments such as RT-PCR,Western blot,TEM test and so on,and the datas show there are similar morphological structures,antigenicity,type specificity between the pseudovirus and the original polio virus.In the second part of the study,polio pseudovirus-based serum neutralization test is researched.But it's impossible to determine serum titer by observation of cytopathies because of pseudovirus' s one-cycle infection.The results of the study show that there is a good linear relationship between the titer of pseudovirus(50~1600 TCID50/50ul)and the luciferase signal RLU after infecting cells.Thus,the serum titers can be determined by the corresponding serum dilution factor which reduced 50% of the positive pseudovirus' s luciferase signal RLU value after neutralization reaction.The pseudovirus-based serum neutralization test was then improved by optimizing the conditions such as the cell density,the detection time and the input of pseudovirus.After that,140 serum samples(48 sIPV,35 OPV,37 wIPV immunizations and 20 negative sera)were evaluated by newly established test,the results showed that there was a good correlation properties with the conventional serum neutralization test.Specifically,the correlation coefficients ranged from 0.8207 to 0.9215(P<0.0001).Thus,the pseudovirus can replace the original virus in the serum neutralization test and it's a simple,efficient safe and specific evaluation method.In addition,the study also carried out high-throughput optimization of the pseudovirus-based serum neutralization test,for instance,the usage of white clear bottom assay plates and the optimization of the proportion of cell lysate and luciferase detection reagent without affecting the test results.As a result,the method is more suitable for high throughput serum neutralization test and greatly improving the efficiency ensuring the experimental results.The packaging of the polio pseudovirus have responded positively to the GPEI plan,and provided a new way to study the polio virus.On the other hand,it solved the problem of evaluating the effectiveness of poliomyelitis vaccine by serum neutralization test after the polio virus sealing and played an important role in the development of the poliomyelitis vaccine. |
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