Establishment Of An Indirect Elisa Method For The Serum Antibodies Detection And Construction Of Recombinant Baculovirus Vaccine Of African Swine Fever Virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) which is an arthropod borne DNA virus of the family Asfarviridae. ASF cause domestic pig and wild boar populations with serious syndrome, such as high fever, extensive haemorrhages and intensive necrosis of lymphoid tissue, which results in great economic losses to the pig industry in most epidemic and new-outbreak areas. Study on the ASFV antibody detection methods and vaccine development is of great significance for preventing ASFV from spreading into our country.In this study, to provide a rapid and effective method for ASFV serum antibodies detection, recombinant VP73protein and standard ASFV-positive serum were applied to develop an indirect ELISA method for detecting ASFV antibodies in large-scale screening of swine serum samples. Meanwhile, the recombinant baculovirus expressing VP73protein of ASFV in mammalian cells was constructed and may be a good candidate in the further development of ASFV vaccine.The main projects in this study are as follows:1. Preparation of VP73protein and monoclonal antibodies of ASFVThe VP73gene of ASFV was synthesized according to the published sequence and then cloned into prokaryotic expression vector pGEX-KG Recombinant plasmid pGEX-KG-VP73was transformed into E.coli BL21(DE3) and induced to express efficiently by IPTG. The western blot using positive anti-ASFV serum as the first antibody demonstrated that the recombinant VP73protein could react with antibodies raised against VP73. The purified VP73fusion protein was used to immunize4-week-old female BALB/c mice to prepare the monoclonal antibodies. After detecting and screening by using indirect enzyme linked immunosorbent assay (ELISA), two hybridoma cell lines stably secreting VP73-specific monoclonal antibodies were obtained by their strong immunoreactivity, named1E6and1H3. Subtypes are IgG2b and IgG1, respectively. The light chains are all Kappa chain. Then specifity of the McAbs was characterized by indirect immunofluorescent assay and western blot. The results showed that the two McAbs were specific to VP73protein. 2. Development and evaluation of an indirect ELISA for the detection of serum antibodies against ASFVA VP73recombinant protein-coated indirect enzyme-linked immunoassay (i-ELISA) for the detection of ASFV serum antibodies was developed using a horseradish peroxidase (HRP)-labeled rabbit anti-pig IgG as secondary antibody. The sensitivity assay showed the maximum standard ASFV-positive serum dilution for detection was2560-fold, which indicated the i-ELISA was capable in detecting ASFV antibody with high sensitivity. The coefficient of variation of each sample was below10%in both of the intra-assay and inter-assay. The OD450values of the standard positive control and the standard negative control were shown with no significant decrease in the tests with well-coated plate placed in37℃for at least5days. Compared with the commercial blocking ELISA kit, the specificity and sensitivity of the i-ELISA were99.1%and94.3%, respectively. There was an excellent agreement between the results obtained by commercial ELISA and the i-ELISA (kappa=0.944).3. Construction of recombinant baculovirus expressing VP73protein of ASFVThe VP73gene was synthesized and cloned into pFastBacl, in which the VSV/G gene is under the control of the polyhedron promoter of pFastBacI and the DNA fragment containing the VP73expression cassette is controlled under the CMV promoter (CMV-VP73-BGH poly(A)). In addition, the VP73protein was expressed in BHK-21cells infected with the recombinant baculovirus (rAcNPV-G-VP73) generated from baculovirus system and identified by western blot. Our results suggested that the recombinant baculovirus expressing VP73protein could be used as a good candidate for further development of vaccine for ASFV.