Prokaryotic Expression And Characterization Of P10 And PS273R Proteins Of African Swine Fever Virus

African swine fever(ASF)is an acute,hot and highly infectious disease of pigs caused by African swine fever virus(ASFV).It is listed as an epidemic disease that must be reported by the World Health Organization and is classified as a class of animal epidemic disease in China.The disease is characterized by a rapid and acute course of disease.Clinically,it often shows fever and bleeding.The disease is highly infectious.It is widely spread through direct or indirect African swine fever virus.Although it is not a zoonotic disease,it will sicken pigs through animal transmission,and the death toll is very high.Once it is found,it will be slaughtered in large numbers.It is an infectious disease that the pig industry cannot avoid.African swine fever virus has a complex icosahedral structure and can encode more than 150 proteins.At present,all the protein functions of acfv have not been fully understood.Exploring the functions and characteristics of the proteins is particularly important for the development of vaccines and the screening of detection targets.K78R gene is located in the pseudonucleus and expresses p10 protein.At present,it is considered that its main function is related to replication.However,whether it has immunogenicity and the response law of the body to p10 protein are not clear.S273 R gene is located in the core-shell and expresses pS273 R protein,which is an enzyme with hydrolysis function.This test mainly focuses on the p10 protein and pS273 R protein encoded by the K78 R and S273 R genes of African swine fever virus.The research contents are as follows:(1)Construction of prokaryotic expression recombinant plasmids of pET-32a-p10 and pET-32a-pS273R:using the whole genome of ASFV SY18 preserved in the laboratory as a template,K78 R and S273 R genes were amplified from them with designed specific primers.After double digestion with empty vector pET-32 a,T4 ligase ligation,they were transformed into E.coli DH5α In the cloned bacteria,the positive clones identified correctly by PCR were sent to Jilin kumei biological Co.,Ltd.for sequencing,the recombinant plasmid was extracted and transformed into BL21(DE3)expressing bacteria.After the optimal induction conditions were determined by pre experiment,a large number of induction was conducted.After splitting the broken bacteria,the fusion protein with his tag was purified by incubation with Ni NTA affinity chromatography column,and identified by SDS-PAGE and Western blot,The specific fusion protein with single band was purified.(2)To establish an indirect ELISA method for the detection of p10 protein antibody: the prokaryotic expression of p10 protein was coated on the ELISA enzyme labeled plate,the immune pig serum was used as the detection sample,the enzyme labeled Sheep anti pig secondary antibody was used as the detection antibody,and the TMB chromogenic solution was used;The reaction conditions with the maximum P/N value were determined through checkerboard verification.The coating antigen concentration was determined to be 2 μg/ml p10 protein.The serum to be tested was diluted 500 times,and the secondary antibody labeled with HRP was diluted 1:15 000 times.It was confirmed that the method had good repeatability,and the same samples were detected with the African swine fever blocking ELISA diagnostic kit of ingenasa company in Spain.The total coincidence rate was 88%.After testing a large number of serum samples,it was found that the antibody value detected by this method was highly consistent with the virus content in infected pigs,that is,the lower the Cq value,the higher the virus content in vivo,and the higher the antibody value detected by p10.Therefore,this method can detect whether domestic pigs produce antibodies against p10 protein through blood collection,so as to know the virus content in pigs.This method provides a new idea for detecting the immune response and infection of pigs.At the same time,we also prepared rabbit anti-p10 and rabbit anti-pS273 R polyclonal antibodies against p10 and pS273 R proteins: the purified p10 and pS273 R proteins were mixed with Freund’s adjuvant and treated with ultrasound to the state of water in oil emulsion,and then immunized New Zealand white rabbits for many times.Blood samples were taken at an interval of one week to prepare polyclonal antibodies.The antibody titers measured by indirect ELISA reached 1:32 000 and 1:64 000 respectively,and were verified by Western blot,It proved that the polyclonal antibody with specificity was successfully prepared.On this basis,two rabbit anti polyclonal antibodies and red fluorescent deletion virus ASFV SY18-△MGF were used for neutralization test.It was found that there was no neutralization effect.In conclusion,through this study,we constructed the prokaryotic expression recombinant plasmids of pET-32a-p10 and pET-32a-pS273 R,established an indirect ELISA antibody detection method using p10 protein as antigen,and prepared Rabbit anti polyclonal antibody,which laid a foundation for exploring its immunogenicity and reactivity,and went further in the direction of establishing an African swine fever detection method and screening vaccine strains.