Screening Of ASFV Immunosuppressive Genes And Preliminary Establishment Of RPA Detection Methods

African swine fever(ASF)is an acute,heat and highly contagious disease caused by African swine fever virus(ASFV)in domestic and wild pigs.ASFV has been spreading in many countries around the world since the first outbroke in 1921.On August 1,2018,the first ASF epidemic was discovered in Shenyang,Liaoning Province,and then it has been spreading in 32 provinces/municipalities/special administrative regions such as Henan,Jiangsu,Zhejiang,Anhui,Heilongjiang,Inner Mongolia,Jilin,Tianjin and Hong Kong,has caused great economic losses.Phylogenetic analysis revealed that the strain belonged to be gene type II,which is the same with the Georgia 2007/1 strain.ASFV genome has 30%multiple-copies in open reading frame which named multigene families(MGFs),and MGF360 genes have been proved to inhibit the IFN-mediated innate immune response.Our previous research found that MGF360-12L,-13L and-14L genes are immune evasion genes which can be one of the main targets for the preparation of gene-deficient vaccines.Therefore,the detection method of MGF360 inhibitory genes are particularly important.Firstly,MGF360-12L,-13L and-14L genes of ASFV Georgia 2007/1 stain were synthesized and cloned into pcDNA3.1 eukaryotic vector.There combinant plasmids were transfected into PK15 and293T cells,and the IFN-?expression levels were verified by real-time PCR.12L and 13L were found to have stronger effection on inhibiting the expression of IFN-?.Subsequently,specific primers were designed for 12L or 13L genes,in order to establish recombinase polymerase amplification(RPA)isothermal detection method.The results showed that 12L-Basic RPA method could achieve stable amplification at 35?in or 30 min,while 13L-RPA used 20 min at 39?.The sensitivity of the method separately reached 10~3 and 10~2copies,which is consistent with the detection limit of common PCR.Besides,the RPA method only specifically amplified ASFV MGF360-12Lor-13L genes,and had no amplification of PEDV,FMDV,CSFV,PRRSV,PRV and PCV-2 genomes.And then ASFV genome was used as a positive sample to the 12L-RPA and 13L-RPA.Although genome was diluted,12L-RPA and13L-RPA could still detect the presence of the target gene,indicating that the detection method has clinical value.ASFV MGF360-12L or-13L genes were separately cloned into E.coli prokaryotic vectors pGEX6p-1,pET-21a,pET-28a and pET-32a in order to proteins expression.Although the conditions have optimized,12L and 13L proteins were also inclusion body.And the inclusion body protein expressed by pET-21a vector could obtain better dissolution.After purified,12L and 13L proteins concentration reached 200-500?g/mL.In summary,based on ASFV MGF360-12L or-13L gene sequences,the RPA methods was established initially,which can rapidly and specifically amplify 12L and 13L,and may provide certain technical support for the differential diagnosis of subsequent 12L and 13L gene deletion vaccines.At the same time,12L and 13L proteins were expressed and successfully purified,which were pre-stored of the subsequent polyclonal antibody preparation,the establishment of indirect ELISA and the functional study of related proteins.