| African swine fever(ASF)is an acute,highly contagious infectious disease with high lethality caused by African swine fever virus(ASFV).ASF is listed as a notifiable disease by the World Organization for Animal Health(OIE).In 1921,ASF was first identified in Kenya and spread throughout sub-Saharan Africa.In August 2018,ASF broke out in China for the first time and quickly swept the country and continued to spread.It has now become the most important infectious disease threatening my country’s pig industry,causing huge economic losses to the pig industry.So far,there are still no effective vaccines and antiviral drugs on the market to prevent and control ASF,and the spread of the epidemic can only be controlled through early detection and early treatment.Therefore,it is necessary to establish a simple,rapid,specific,and large-scale on-site detection method.ASFV is an icosahedral particle composed of five independent structures.The genome is 170-194 kb long.It is a double-stranded closed linear DNA molecule containing 151-171 open reading frames.The function of almost half of the ASFV genes is currently unknown.The study found that the main capsid protein p72 encoded by the viral gene B646 L accounts for about 1/3 of the total viral particles,and its amino acid sequence is very conservative in different ASFV strains.The structural elucidation of p72 showed that the three p72 monomers form stable trimers in the native state.p B602 L is a non-structural protein encoded by the ASFV gene B602 L,which can assist p72 to fold correctly to form trimers,and plays an important role in the assembly of ASFV icosahedrons.In addition,previous studies have confirmed that the p72 antibody is produced at an early time and at a high level in animals infected with ASFV,which is an ideal target for establishing antibody detection methods.p B602 L is a non-structural protein of the virus.The antigenic epitope is conserved and has good antigenicity.The antibody level in the serum after infection is high and lasts for a long time.It can be used as an effective target for the detection of antibodies for persistent virus infection.Therefore,in this study,a colloidal gold immunochromatographic test strip was established based on the recombinant protein p72 to provide a simple and rapid monitoring and detection tool for the effective prevention and control of ASF;An indirect enzyme-linked immunosorbent assay(ELISA)antibody detection method was established based on the recombinant protein p B602 L.It can perform quantitative and qualitative analysis of large-scale samples.After the successful development of ASF vaccine in the future,this method can also be used to distinguish wild virus infection from vaccine immunity.The details are as follows:1.Development and application of colloidal gold immunochromatographic test strip based on recombinant protein p72.In this study,the ASFV gene sequence(Gen Bank: MK333180.1)was obtained from NCBI,and the p72 and p B602 L gene sequences were codon-optimized and synthesized.They were constructed on p CMV vectors and named as p CMV-p72 and p CMV-p B602 L.The p CMV-p72 and p CMV-p B602 L plasmids were co-transfected into HEK293 cells,and the p72 protein was successfully expressed.The recombinant protein p72 was purified by Flag antibody affinity chromatography,and the p72 trimer was further separated and purified by gel filtration chromatography.Finally,a correctly folded and high-purity p72 trimer was obtained.Colloidal gold was used to label the p72 trimer,Staphylococcal protein A(SPA)was used as the detection line to capture the protein,and anti-p72 protein polyclonal antibody was used as the quality control line to develope colloidal gold immunochromatographic test strip which with high detection sensitivity(the lowest detection limit of ASFV antibody-positive standard serum can reach 1:204,800),good repeatability(the coefficient of variation between test strips is less than 15%),and good specificity.In the absence of vaccines,the presence of antibodies is an important indicator of viral infection,and this test strip can be used for ASFV infection monitoring.2.Development of an indirect ELISA antibody detection method based on recombinant protein pB602 L.The recombinant plasmid p CMV-B602 L was transfected into HEK293 F cells to obtain the recombinant protein p B602 L with high biological activity and high expression.The high-purity target protein was prepared by two-step Ni affinity chromatography and gel filtration chromatography.Serological tests are mainly the detection of specific antibodies.Because of its easy qualitative and quantitative detection,easy standardization and high sensitivity,ELISA detection method is very suitable for dynamic monitoring of pig herd health and large-scale sample epidemiological investigation.It is the first antibody detection method recommended by OIE.Compared with commercial ELISA detection kits,the detection method established based on recombinant protein p B602 L has higher sensitivity and the coincidence rate can reach 93.75%.The repeatability between batches and within batches is good,and the coefficient of variation(CV)is lower than 10%,with good stability.It is suitable for serological monitoring of ASF.In conclusion,this study successfully expressed the recombinant proteins p72 trimer and p B602 L by using the eukaryotic expression system mammalian cell HEK293,which provided abundant experimental materials for further functional studies of ASFV p B602 L and p72 proteins.In addition,based on the p72 trimer,ASFV antibody detection colloidal gold immunochromatographic test paper was developed and pilot-scale production was carried out,and clinical experimental application was carried out in pig farms.An indirect ELISA assay was successfully established and evaluated based on the viral nonstructural protein p B602 L.The two antibody detection methods established in this study provide effective tools for rapid on-site detection of ASFV in pig farms,and provide important technical support for ASF prevention and control work and epidemiological investigation. |
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