Genome Sequencing And Analysis Of Pig Jev Guangxi Isolate Strain Fc792, Establishing The Method Of The Rt-Lamp, And Prokaryotic Expression The E Gene Of FC792Strain

Japanese encephalitis (JE) is acute infections disease, which caused by JEV, and it’s one of most important desease for both human and livestock. Mainly occurs in the Eastern and southEastern of Asia, Australia and other regions and countries, its mortality rated up to20%. According to the World Health Organization, the number of JE incidence had reached to35000each year, among them China accounted for80%. JEV can infect pigs, causing the abortion of pregnant sows, boars testicular swelling and piglets nervous system disease, so as caused huge losses to the pig industry.Pigs are the main storage and proliferation host for JEV, so there has important public health significance to diagnosis and control of pigs infected with JEV. This study, used the JEV SA14strain’s genome sequence as template design JEV specificity primers, divided seven-segment amplification of JEV genome sequence of FC792isolate strain, after sequencing and splicing, got the full genome sequence of FC792.The full-length of FC792has10977nucleotides,5’noncoding region include95nucleotides,3’noncoding region has586nucleotides, an open reading frame containing10,296nucleotides, which encodes3432amino acids. The nucleotides and amino sequence analysis revealed that FC792compare with the broad and domestic JEV representatives stains, it’s nucleotides homology shared84.6%to99.7%, amino acid homology were96.2%to99.4%. There were a99.7%and99.6%homology of the nucleotide,99.4%and99.2%homology of the amino acid between SA14-14-2and YUNNAN0901strains, respectively. Phylogenetic analysis showed that the FC792strain belonged to genotype III type, and the phylogenetic relations was the most closest to the SA14-14-2and YUNNAN0901strains. The nucleotides was only0.45%, amino acid was0.76%, the structural protein containing five amino acid differences between the FC792and YUNNAN0901strains.Established the detection method of JEV RT-LAMP, and optimized the system and the conditions of reaction, the sensitivity experiments showed that the detection limit of the JEV RT-LAMP method which established in this study was0.5pg, the specific experiments proved it had high specificity. After the end of the RT-LAMP reaction, by adding SYBR Green I to visualize observation, greatly reducing the detection time.Cloned the JEV FC792strain’s E gene fragments Ea, which containing the antigenic domain Ⅰand Ⅱ; was successfully constructed the prokaryotic expression plasmid of pET-32a-Ea. The Western-blot analysis was used the JEV positive serum of swine as the primary antibody, initially showed that the recombinant protein Ea had antigenicity; purification refolding of recombinant protein Ea, immunized the mouse twice interval one week, eyes blood to separate serum a week after the second immunization.Was positive that tested by the ELISA kit which packeted with JEV, indicating that Ea has antigenicity; used the positive mouse’s serum which detected by ELISA to do the virus neutralization test, and the results showed that the serum of mice immunization with the ability of neutralizing JEV, and the neutralizing antibody titer was1:70, demonstrate that the recombinant protein Ea has good immunogenicity.