Expression Of SFTSV Glycoprotein And N Protein In Eukayotic Cells And Analysis The Biological Activities Of The Recombinant Proteins

Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) is a newly discovered bunyavirus that causes an emerging hemorrhagic fever in rural areas of China. The annual incidence of the SFTS is about five over one hundred thousand in the rural population and the fatality rate is 12% on average. The patients of SFTS are mainly elder people. Since SFTSV was found in China, SFTSV has been reported in South Korea and Japan, and a similar virus has been reported in the United States. SFTSV is a member of the genus Phlebovirus, in the family Bunyaviridae. The genome of SFTSV consists of three RNA segments:the large (L), medium (M), and small (S) segments.The L segment encodes RNA polymerase; the M segment encodes glycoprotein (Gn and Gc) precursor; the S segment encodes two proteins, the N and NSs proteins, in opposite orientations. The Gn and Gc are most likely to induce the production of neutralizing antibodies. A monoclonal antibody(MAb4-5) to the Gn protein has been demonstrated to be a neutralizing antibody.The S segment is the most conservative sequence among the three segments, and the nucleoprotein is the most easily to express in large quantity, which has been tested for immunological diagnsosis of SFTSV infection.Based on the current knowledge, this study aims to construct eukaryotic expression vectors of Gn, Gc NP and the segmented protein of the M segment, and analyze the biological function of the proteins in eukayotic host cells. This research has established the foundation for further rearch on neutralizing antibody and immunodiagnosis of SFTS with recombinant proteins.OBJECTIVE1. Construct the eukaryotic expression vectors of Gn, Gc and NP.2. Construct the eukaryotic expression vectors of overlaped fragments of M segment.3. Express NP and analyze the biological activities of the recombinant NP.METHODS1. Design primers to amplify NP, Gn, and Gc genes.2. Design primers to amplify the M segment into 20 overlap fragments (M1-M20) with the adjacent segments overlapping 30 bp.3. Design primers to amplify the NP, Gn, and Ml genes and to construct GFP fusion protein of each protein for expressing of NP+GFP, Gn+GFP and M1+GFP.RESULTS1. The nucleoprotein NP, glycoproteins Gn, Gc overlapeed M segments (M1-M20) and the fusion proteins of NP+GFP, Gn+GFP, M1+GFP expression vectors were constructed and the recombianant proteins were expressed in Vero cells.2. There is no statistical differences between the expressed NP coated plates and the commercial ELISA-kits.3. Antibodies to NP were genearated by immunizing BALB/c mice with recombinant NP.CONCLUSION1. The eukaryotic expression vectors constructed by pVAXl can express NP, Gn, Gc, M1-M20, respectively after transfected into Vero cells, but the yields of proteins expressed by overlapped M fragment were low.2. The NP in vector pVAXl was expressed well in Vero cells, and the recombinant NP can be used for diagnosis of SFTSV infection with ELISA.