The Study On Detecting Methods Of K205R Gene Of African Swine Fever Virus

African swine fever(ASF)is an acute and fulminating infectious disease caused by African swine fever viru(sASFV), there is currently no vaccine and medicinal prevention and cure available for ASFV . In order to meet the demand of detecting ASFV, K205R gene was amplified from ASFV genomic DNA by designing primers according to the sequence released in GenBank. A real-time quantitative PCR(qPCR)assay for the detection of ASFV based on K205R has been developed by designing the primers and TaqMan probe. The reaction solutions and conditions were optimized by using different concentrations of the primers and Mg2+ and different annealing temperature. The qPCR detective method of ASFV based on p72 recommended by OIE was selected to be as evaluation standards. Evaluation result indicated that two methods shows high specificity and sensitivity, and they could detect 101 and 102 copies of DNA templates in the format of recombinants plasmid and mimicry samples respectively, while qPCR based on K205R had a higher amplification efficiency than qPCR based on p72 when used to detect ASF mimicry samples(99.8%/95%), and could be applied to detect animals and related products.K205R was ligated to expression vector pQE30 and transformed into Escherichia coli, and induced to express by inducing with IPTG. Results indicated that the fusion protein was expressed as a soluble protein identified by SDS-PAGE and Western Blot analysis. Indirect ELISA method was chosen to examine the reactogenicity of the expressed antigen and results showed that the positive antisera could be recognized when diluted up to 1:12800. When the purified fusion protein was used to immunize the Balb/c mouse for 3 times to assess the immunogenicity of pK205R, the mouse sera antibody titer reached over 1:102400, these results had laid the foundation for the serological assay detecting ASFV.