Prokaryotic expression, purification and antigenicity analysis of African swine fever virus pK205R protein

Abstract

African swine fever is an acute, febrile and highly virulent porcine disease causing serious economic losses worldwide. The pK205R protein of the African swine fever virus (ASFV) is largely expressed in the early stages of infection, which has given the K205R gene extensive attention. In this study, the ASFV K205R was cloned and expressed in Escherichia coli BL21 (DE3). Expression of histidine-tagged pK205R with a molecular mass of 44 kDa was determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Optimisation of culture conditions allowed induction of the recombinant protein with 0.4 mM Isopropyl β-D-thiogalactoside (IPTG) at 37 o C for 2 h. The protein existed in cellular supernatant and was purified using a Ni-NTA resin column. The purified protein was used to immunize rabbits four times to enable the production of polyclonal antibodies, and the antiserum titre was detected by ELISA. The results showed that the purified pK205R can react with ASFV positive serum specifically by Western blotting. The pK205R had high antigenicity, which indicated that pK205R could be used as an antigen for detection of ASFV-specific antibodies in ELISA testing, and the recombinant protein could contribute to further research of the action and structure of pK205R.

African Swine Fever Virus K205R Induces ER Stress and Consequently Activates Autophagy and the NF-κB Signaling Pathway

“…Adenovirus-vectored ASFV K205R elicits robust immune responses in swine [17]. ASFV K205R has high antigenicity and can be recognized by hyperimmune antisera from infected pigs, suggesting that K205R has the potential to be used for the detection of ASFV-specific antibodies [18]. In the present study, we report that ASFV K205R activates autophagy and the inflammatory response through triggering ER stress.…”

“…K205R has been shown to have high antigenicity and can be used for the detection of ASFV-specific antibodies [18]. Adenovirus-vectored K205R has been found to elicit robust immune responses in swine, suggesting that K205R may be an effective component of a prototype vaccine [17].…”

A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R

“…Will can it effectively resist the infection of ASFV? Some studies have shown that the pK205R of ASFV has good immunogenicity and is a good antigen candidate for early diagnosis ( 21 ). In this study, pK205R of ASFV was selected as the foreign target antigen and was inserted into the live vector of PRRSV.…”

“…ORF K205R encodes pK205R, an early viral protein that contains a conserved amino acid sequence and is expressed in the early stages of infection from 4 h post-infection (hpi). Prokaryotic pK205R has high antigenicity and can be recognized by hyperimmune antisera from infected pigs in enzyme-linked immunosorbent assay (ELISA) testing, suggesting that pK205R has the potential to be used for the detection of ASFV-specific antibodies ( 18 , 20 , 21 ). Consistently high serological responses against pK205R were observed at late stages of infection compared with the response to structural proteins in domestic pigs and bushpigs infected with a range of different viral isolates.…”

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies

“…In infected macrophages, more than 100 virus-induced proteins have been identified, of which at least 50 can interact with infected pigs or recovered pigs of serum responses, 40 were able to bind to virions. K205R protein can induce a strong immune response in pigs, which is one of the main antigens of ASFV [ 22 , 23 ]. As a protein expressed in the early stages of viral infection, K205R appears at as early as 4 h after infection, and localizes to the ASFV replication factories [ 2 , 24 ].…”