Sftsv Genome Sequencing And Prokaryotic Expression Of Its Nucleocapsid Protein

Since Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) have been reported2011, the epidemic areas were expanded in our country. This virus cause a disease named Severe fever with thrombocytopenia syndrome(SFTS). The main clinical feature include fever, thrombocytopenia and severe multiple organ failure then causing death. Due to the high susceptibility of SFTS and studies for SFTSV is also in its infancy, there is no effective prevention and treatment measures. The research of SFTSV has great significance for the prevention and control of SFTS outbreak.The main purpose of this study was to establish the Real-time fluorescence quantitative RT-PCR detection methods for SFTSV. Isolated SFTSV from the positive serum samples of patient in Zhejiang province. Sequencing the whole genome and analysis the molecular phylogenetic and molecular epidemiological to preliminary understanding the evolution of the Zhejiang region SFTSV regional characteristics. After SFTSV once infected the host, the antibodies of the nucleocapsid (N) protein can be quickly induced. Therefore, we cloned the N protein gene of SFTSV. The recombinant expression plasmid was prokaryotic expressed. And the recombinant protein was purified and identified. The purified recombinant N protein, which we expect it to be immunological activity, can be used for clinical diagnosis, or provide an important antigen for polyclonal or monoclonal antibodies. It also laid the foundation for SFTSV preliminary serological detection methods and the development of SFTSV engineering vaccine. The main findings are as follows:The TagMan real-time quantitative RT-PCR detection method for SFTSV was established, using the specific primers and probe designed from conserved regions of the gene RdRP. the method to isolate the SFTSV from Vero using virus culture was established. The SFTSV Zhejiang strain Zhejiang/01/2011which has been isolated was identified by quantitative real-time RT-PCR, and its viral RNA was extracted.17overlapping fragments covering the whole genome were amplified by RT-PCR. And the entire genomes were formed by sequencing and assembly the fragments. The results showed that SFTSV Zhejiang strain genome cDNA sequence has3fragment. The S segment contains1745nucleotides. The M fragment contains3378nucleotides, and the L segment contains6368nucleotides. Since these primers was located in relatively conservative area of the viral genome, they can be used for sequencing the whole genome of other SFTSV. The sequence of virus strain Zhejiang/01/2011has been uploaded to GenBank. The accession number of S fragment is KJ597823. The accession number of M fragment is KJ597824. The accession number of L fragment is KJ597825. The SFTSV sequence of Zhejiang strain was compared with the sequences of SFTSV that have been downloaded from GenBank database and the sequences of strains of the genus phlebovirus in the Bunyaviridae family to generate the phylogenetic tree and analysis the homology using MEGA and RAxML software. Sequence analysis showed that Zhejiang/01/2011was in the same branch with the strains that isolated from Japan, and had higher similarity. Especially had the highest similarity with Japan/SPL004A/2013strain. The similarity of the S fragment was98%. The similarity of the M fragment was97%. And it was98%that of the L fragment. Zhejiang/01/2011was not in the same branch with the strains that isolated from the rest regions of China, and had higher difference. Meanwhile, the results also confirmed that the virus strain belongs to genus phlebovirus.The gene of N protein (738bp) which located in the segment S of SFTSV was successfully amplified by RT-PCR. The cloning vector pMD-19T-SFTSV-NP and the prokaryotic expression vector pET-42a (+)-SFTSV-NP were constructed and analyzed by sequencing. The prokaryotic expression vector pET-42a(+)-SFTSV-NP was transformed into E.coli BL21(DE3). The IPTG induction system was optimized, and the recombinant protein was highly expressed. SDS-PAGE result showed that the recombinant protein mainly expressed as inclusion body protein. The recombinant N protein was purified by Ni-chelating affinity chromatography. The result of Western blot showed that the recombinant protein had good antigenicity, and it laid the foundation for the early biochemical diagnosis of SFTSV.