Screening Of K205R-Specific Nanobodies For ASFV Non-Structural Protein And Establishment Of ELISA Detection

African swine fever(ASF)is a highly contagious and deadly viral disease of domestic pigs caused by the African swine fever virus(ASFV),which is a notifiable infectious disease of the World Organization for Animal Health(OIE).In 2018,Shenyang City,Liaoning Province,China,reported the first case of African swine fever infection with highly virulent gene type II,and since then it has spread throughout the country at a very fast speed,causing the number of pigs to plummet and the price of pork to rise rapidly,causing great economic losses to the pig industry and affecting the normal life of the people.At present,there is no effective vaccine or drug against this virus,ASFV has become one of the difficult viruses to overcome,the current ASF prevention and control is mainly culling and strict disinfection measures,early detection and control of ASF epidemics from the source is the main prevention and control measures,therefore,the establishment of a rapid time-saving and responsive early detection method of ASFV is crucial.K205 R is a non-structural protein expressed in the early stage of ASFV infection,which has good antigenicity in the diagnosis of ASFV infection throughout the period,and can induce the production of specific serum antibodies in pigs,which is an ideal target protein for the establishment of ASFV serological detection methods.Nanobody(Nb)is an antibody fragment existing in camelids,containing only heavy chain Ig G antibodies,with the advantages of small molecular weight,simple structure,high antigen binding affinity,etc.,and is widely used in the field of disease diagnosis and treatment,however,nanobodies are rarely reported in the diagnosis and treatment of animal diseases.In this study,the purified K205 R protein immune male Bactrian camel was successfully screened for 5 specific nanobodies against K205 R protein by phage display technology,and a platform for simple operation and rapid expression of Nbs-HRP fusion protein was constructed,and a rapid time-saving and highly sensitive c ELISA detection method was developed by using fusion protein as a detection probe,the main contents and main results of this study are as follows:1.Expression and purification of ASFV-K205 R proteinThe successfully constructed recombinant plasmid p ET-30a-ASFV-K205 R recombinant plasmid was converted into Transetta(DE3)expression competent cells,and the protein expressed in the form of inclusion bodies was obtained after IPTG induction expression,and after Ni-NTA purification,a high concentration of K205 R recombinant protein was obtained,which was used for the immunity of Bactrian camels.2.Construction of VHH phage library and screening of ASFV K205 R protein specific nanoantibodiesAfter the fifth immunization,Bactrian camels were collected blood,the total RNA of peripheral blood lymphocytes was extracted and reverse transcribed,and the VHH gene was amplified using nested PCR as a template,inserted into the p CANTAB 5E bacteriophage vector,electroporated to TG1 competent cells,and finally screened 5 specific nanobodies against K205 R protein through 3 rounds of rescue and biological panning,and the analysis of ELISA detection results showed that Nb1,Nb35 and Nb82 had the strongest binding power.3.Establishment and evaluation of competitive ELISA assayNbs were cloned into p CAGGS vectors,recombinant plasmids of eukaryotic expression vectors were transfected with HEK293 T cells,and nanobodies secreted expressed in cell supernatants were collected.The ELISA detection results showed that the three nanobodies were able to block the reaction of K205 R protein with positive serology,among which the blocking rate of Nb1 was high,Nb1 was selected as the detection probe to establish a competitive ELISA detection method,and the reaction conditions were optimized,and the Cut-off value was determined by using the established c ELISA detection method to be 34.8%,with high specificity and sensitivity of 100%,and the intraplate coefficient of variation was 2.23%-8.45%,and the median value was 5.34%.The interplate coefficient of variation was 3.71%-10.9%,the median value was 7.31%,and the compliance rate with commercial kits was99.3%.In summary,five specific nanobodies against ASFV K205 R protein were screened in this project,and finally Nb1 was used as the detection probe to establish a competitive ELISA detection method for rapid detection of porcine serum antibodies,which has the advantages of low production cost,simple operation and short time-consuming,and has broad application prospects in the detection of ASFV antibody in clinical porcine serum.