A Serological Diagnostic Method For African Swine Fever Based On Luciferase Immunoprecipitation System

African swine fever(ASF)which is a highly contagious swine disease caused by the African swine fever virus(ASFV)has caused significant economic loss to the swine industry worldwide.The ASFV strains circulating in China have shifted from the initial virulent genotype Ⅱ strains to more diverse field strains.Although the pathogenicity of ASFV field strains may be significantly reduced,their horizontal transmission ability should not be overlooked.To date,no ASF vaccine has been approved officially in China.The emergence of novel recombinant strains and the illegally used vaccine strain poses more challenges to the elimination of ASFV in China.The most effective measures of ASF control are restricted biosafety measures in farms,accurate and fast diagnosis,and elimination of infected animals.Therefore,it is of great practical significance to develop fast and accurate serological diagnostic methods.To date,several serological diagnostic methods are available for the African swine fever virus,including different types of ELISA,colloidal gold immunochromatographic test strips,Western blot,etc.These detection methods have some shortcomings,such as being time-consuming,nonspecificity issues,and high requirements for detection equipments and experimental conditions.Here,we developed and optimized a luciferase immunoprecipitation system(LIPS)for ASF serological diagnosis.Initially,we established a polyclonal HEK-293T cell line by lentivirus transduction for the production of GLuc-p30 antigen.A lentiviral plasmid carrying the codon-optimized coding sequence of ASFV p30 was cloned into the was constructed using the lentiviral vector pLVX-CMV.The lentivirus expressing GLuc-p30 was generated by DNA transfection of HEK-293T cells with pLVX-CMV-GLuc-p3 0 and two package plasmids,pCMV-VSV-G and psPAX2.HEK-293T cells transduced with this lentivirus expressing GLuc-p30 were further selected with puromycin.In addition,the lentivirus also contains an expression cassette of a green fluorescent protein.Based on puromycin selection and green fluorescence signal screening,we successfully established a polyclonal HEK-293T cell line expressing GLuc-p30.The genetic stability of this cell line was further confirmed by serial passage,and the guassia luciferase assay was performed to monitor the expression of GLuc-p30 secreted into the culture supernatant.For all passages,the expression levels of GLuc-p30 were around 2x107 relative luciferase units/20 μL culture supernatant.With the GLuc-p30 antigen,we established a LIPS assay for the detection of ASFV antibody responses in pig serum samples.Subsequently,we optimized the reaction conditions of LIPS,including coating conditions,blocking conditions,incubation conditions,and dose of recombinant antigen per reaction.We also generated the Nluc-p30 antigen for LIPS detection of ASFV antibody response.In comparison with widely used indirect ELISA-based p30,our LIPS assays exhibited comparative diagnostic specificity and better diagnostic sensitivity.Therefore,we established and systematically optimized a LIPS-based serological diagnostic method for ASFV infection.Our LIPS assays for ASFV serological diagnosis will be promising methods for ASFV diagnosis and may have a broad market prospect.