Study On The Construction And Biological Detection Of Poliomyelitis Pseudovirus Sabin Strains

Poliovirus,abbreviated as poliovirus,belongs to the Picomaviridae family,is a nonenveloped single-strand sensitive RNA virus.The epidemic of poliomyelitis was effectively controlled by vaccine inoculation.After poliovirus eradication,all live viruses including attenuated strains will be sealed and preserved.It is of great value to explore the evaluation of human immunity with polio pseudo virus in the post polio era.In this study,a method for the construction of Sabin II type pseudo virus particles PVS2 was established,and the biological characteristics of PVS2 were studied to determine its effectiveness in the following experiment,which established the foundation for neutralization experiments with pesudovirus.Firstly,the pcDNA-P 1-S2 plasmid expressing the capsid protein and the skeleton plasmid pRepGL containing the reporter gene were constructed by homologous recombination.Then the pseudo virus was prepared by in vivo and in vitro synthesis of Replicon:successively transfection of pcDNA-P 1-S2 and Replicon RNA which transcripted by pRepGL in vitro;co-transfection of pcDNA-P 1-S2,pRepGL and plasmid pCAG-T7-puro which expressing T7 RNA polymerase.There was no significant difference in PVS2 luciferase activity between these two methods.We explored the PVS2 preparation way from 4 aspects From the following 4 aspects:transfection reagent,medium composition,293T cell age and plasmid proportion,and then the luciferase activity of PVS2 can be up to 1.2× 105 RLU.The harvested pseudo virus PVS2 was identified to possess similar morphological structure to the original virus with serum specificity to conjugate neutralizing antibody effectively.The pseudo virus titer and neutralization titer could be determine through the activity of fluorescent protein and luciferase.Meanwhile the sensitivity of luciferase assay was high.The titer of PVS2 was 4 Lg IU/mL which determined with luciferase.At the same time,Sabin type II replicon was used as parental material to recombine Sabin type I and type III capsid protein P1,the recombinant peseudovirus has a serum specificitywas identified by different types of antibodies and the result showed that sera specificity.In conclusion,this study explored the transfection conditions and first established the preparation method of Sabin Strain pseudovirus with Replicon RNA transcript in vivo.The morphology,serology and antigenicity of the Sabin pseudo virus particle PVS2 were similar to that of the Sabin strain.Recombinant I,III type pseudovirus particles Re-PVS1 and Re-PVS3 were obtained with the genetic resortment with type II Replicon and Sabin I,III type capsid protein gene P1.