Construction Of The Minigenome System For Severe Fever With Thrombocytopenia Syndrome Bunyavirus

Background:Severe fever with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome, SFTS) is a severe fever with thrombocytopenia as the main characteristics of the new infectious diseases. The pathogen of severe fever with thrombocytopenia syndrome virus (Severe fever with thrombocytopenia syndrome bunya virus, SFTSV), is a new tick-borne arbovirus. The virus was first discovered in rural areas of Hubei, Henan province and other places in China in2009. SFTSV can cause fever, gastrointestinal symptoms, symptoms of thrombocytopenia, and leucopenia, and multiple organ failures are resulting in death. This new infectious disease mortality was12%on average, regional mortality rates as high as30%. Since the virus was first discovered in the areas of central China in2009, has again frequently occurred in the areas of central and northeast China in2010. Owing to the lack of effective resistance fever with thrombocytopenia syndrome virus drugs and vaccines, so the virus still exist a serious threat to the health of human beings.Purpose:The experiment study of viral gene function, transcription and replication process by constructing SFTSV minigenome. Owing to SFTSV transcription or copy required a lot of original, this experiment also improves the transfection efficiency by constructing the method of cell lines, increase the possibility of successful minigenome. SFTSV structural proteins were expressed for antibody preparation. This experiment will use reverse genetic technology method for the research of the virus, lay the foundation for future drug screening and the vaccine production work.Method:1. Construction of minigenome:We were builded respectively with SFTSV S, M, L, the section5’and3’UTR area and insert in the middle report gene egfp recombinant plasmid. Next, the above recombinant plasmid respectively with NP and RdRp protein expression of recombinant plasmid (previous constructed) combination transfection mammalian cells, evaluate the success and efficiency of minigenome by report the differences in the level of the gene egfp expression.2. Stabilized express SFTSV NP protein cell line screening:We were used G418to evaluate the toxicity experiment of293cells, select the appropriate concentration of G418that used for the screening of subsequent cell line. Then we transfected SFTSV NP and the recombinant plasmid into293cells with G418resistance gene in order to select stabilized express protein cell lines by dilution method. Therefore, identification of this cell lines.3. Prokaryotic expression of SFTSV Gn, Gc and RdRp protein:We designed the corresponding primers to build the various protein prokaryotic expressive recombinant plasmid. Then turn positive recombinant plasmid of Escherichia coli, a small number of induced proteins.Consequence:1. The recombinant plasmids with the inserted report gene egfp were obtained.2. Recombinant plasmids with the report gene egfp and expressive NP and RdRp protein recombinant plasmids pcDNA-NP/L were cotransfected into mammalian cells. Then the reported gene egfp was expressed green fluorescence.3. The SFTSV NP-293cell line which was expressed SFSTV NP protein has been obtained.4. SFTSV structural proteins prokaryotic expressive were obtained.Conclusion:The minigenome of severe fever with thrombocytopenia syndrome bunya virus (SFTSV) is preliminary established. Therefore, the SFTSV NP-293cell line which was expressed SFSTV NP protein can improve transfection efficiency, and SFTSV structural proteins were prokaryotic expressed in E.coli.