Expression Of ASFV P30 And P72 Proteins And Establishment And Application Of Indirect ELISA Antibody Detection Method

African Swine Fever(ASF)is an acute,febrile,highly contagious animal disease caused by African Swine Fever Virus(ASFV).Pigs of all breeds and ages can be infected with the disease.The virus has a complex structure and can encode over150 viral proteins,including over 50 structural proteins.P30 protein is a kind of envelope protein,which is expressed in the early stage of virus infection and participates in the process of virus internalization and virus invasion into host cells;P72 protein is the main capsid protein of the virus,accounting for 32% of the viral protein,and is expressed in the late stage of virus infection.Due to the current lack of effective ASF vaccines,early pig testing and biosafety prevention and control are still the main means of preventing and controlling ASF.The detection method of ELISA antibody level can accurately and timely detect the level of virus antibody in pigs,judge the infection status of pigs,and contribute to more effective prevention and control of African swine fever.Using the African classical swine fever virus ANHUI strain included in the Gen Bank database,the gene sequence encoding the P30 protein and the P72 truncated fragment containing the P72 protein neutralizing antigen epitope were amplified.Through double enzyme digestion and T4-DNA ligase ligation,the recombinant plasmids for prokaryotic expression of p ET-32a-P30 and p ET-28a-P72 were successfully constructed,and finally transferred to Rosetta expression bacteria.The protein expression was determined through preliminary experiments and the protein expression conditions were gradually optimized.After inducing a large number of expressed proteins in the cell and crushing the cell by ultrasound,the target protein with His label was incubated and purified using a Ni-NTA affinity chromatography column.Finally,the protein purification results were verified by SDS-PAGE and Western-blot.The polyclonal antibodies against P30 and P72 proteins expressed in Test 1 were prepared by immunizing New Zealand white rabbits at time intervals,and two eukaryotic expression vectors,p CMV-P30 and p CMV-P72,were constructed.The eukaryotic expression plasmids were transfected into 293 T cells,respectively.The specificity and sensitivity of the two rabbit antiseras were verified by IFA test and Western-blot using two rabbit antiseras as primary antibodies.An indirect ELISA antibody detection method for P30 and P72 proteins was established.The results showed that in the P30 protein detection method,the P30 protein was diluted to 0.5 using a coating solution μg/m L,wrapped overnight at 4 ℃.5% skimmed milk was sealed at 37 ℃ for 3 hours,the first antiserum was diluted at1:800 and then added to the enzyme labeled well.Incubated at 37 ℃ for 30 minutes,the second enzyme labeled antibody was diluted at 1:10000,incubated at 37 ℃ for 30 minutes,and treated with TMB chromogenic solution for 10 minutes.Then,the termination solution was added at 50 μL/well and the OD450 value was read;In the P72 protein detection method,dilute the P72 protein to 4 with the coating liquidμg/m L,wrapped overnight at 4 ℃.5% skimmed milk was sealed at 37 ℃ for 3 hours,the first antiserum was diluted at 1:200,incubated at 37 ℃ for 30 minutes,the second enzyme-labeled antibody was diluted at 1:8000,incubated at 37 ℃ for 30 minutes,and treated with TMB chromogenic solution for 10 minutes.Then,a termination solution was added at 50 μL/well and the OD450 value was read;The determination of critical values,as well as the verification of specificity and repeatability,respectively,showed that the two detection methods were good.Two methods were used to detect180 clinical pig sera,and compared with the results of the commercial African swine fever indirect ELISA antibody detection kit,the P30 coincidence rate was 97.8%,and the P72 coincidence rate was 97.3%.It has been proved that the two methods can be initially applied to the clinical serum detection of African swine fever,and can quickly diagnose the early and late antibody detection of ASFV.It is of great significance to master the ASF antibody level in the pig population and understand the infection status of ASF in the pig population in the later stage.It also provides a reference for the development of a non plague antibody ELISA diagnostic kit for the later establishment of P30 and P72 fusion proteins.