Characteristic Analysis Of The Whole Gene Sequence Of SFTSV And Preparation Of Polyclonal Antibody Against Nucleoprotein

SFTS is a new acute infectious disease that is infected by the new Bunia virus(SFTSV)and transmitted by the bite of a tick.The typical clinical symptoms of the disease mainly include severe fever,thrombocytopenia and leukopenia,and the mortality rate is high.The terrain of Northeast China is mountainous,rich in biological resources,complex geography and variability in climate,resulting in abundance and diversity of species,providing an ecological and biological basis for the survival and reproduction of ticks.The number of cases of the disease has been increasing year by year in recent years,posing a huge threat to the health of the population,especially the farmers.However,the disease currently mainly relies on epidemiological data analysis,clinical symptom diagnosis and blood indicators,and it is easily interfered by other fever diseases and tick-borne diseases,which may cause delays or even misdiagnosis.In addition,there is a lack of basic research on the origin and evolution of the pathogen.In this study,molecular biological diagnosis was performed on the blood of patients,the entire sequence of neobunya virus was amplified,and phylogenetic tree analysis was performed.Collect ticks distributed in the patient's residence for pathogen analysis.Select one of the positive samples as a template,amplify the SFTSV-NP sequence,obtain the recombinant protein through the E.coli prokaryotic expression system,and use SDS-PAGE and Western Blot methods to determine and purify the target protein.The purified protein was used as an immunogen to immunize experimental animals to obtain a polyclonal antibody,which was identified by Western Blot and ELISA test to prove whether the polyclonal antibody is immunogenic.The main results of the experiment are as follows:(1)17 suspected SFTS patients were tested by molecular biology,and 5 of them were diagnosed as positive for SFTSV.The whole genome sequence of SFTSV was determined,and two whole genome sequences were obtained.The ticks collected from the patient's place of residence and adjacent areas were determined by the SFTSV whole genome sequence,and two complete sequences were obtained.The results showed that they were all genotype A.Two strains of SFTSV isolated from patients and two strains isolated from ticks The gene sequence of SFTSV is highly consistent,which proves that ticks are the source of transmission of SFTSV;(2)According to the SFTSV-NP sequence,the prokaryotic expression primers are designed to amplify a 738 bp size fragment.The recombinant protein was obtained through the E.coli prokaryotic expression system,and the nucleoprotein was purified by affinity chromatography separation technology after experimental verification,and a large number of high-concentration and high-purity proteins were obtained through electro-white gel electrophoresis and western blotting;(3))The target protein is used as an immunogen to prepare polyclonal antibodies,which are verified by ELISA and Western Blot experiments to provide detection technology for the early and rapid diagnosis of the disease.This experiment has identified the immunogenicity of SFTSV-NP from molecular biology and serological studies,and has a further understanding and understanding of neobunya virus,laying a foundation for the establishment of a commercial ELISA kit;for further research on its cause Establish a preliminary basis for disease mechanisms and control measures.