Self-assembly into virus–like particles of the recombinant capsid protein of porcine circovirus type 3 and its application on antibodies detection

Abstract

PCV3 capsid protein (Cap) is an important antigen for diagnosis and vaccine development. To achieve high-level expression of recombinant PCV3 Cap in Escherichia coli (E. coli), the gene of wild-type entire Cap (wt-eCap) was amplified from clinical samples, and three optimized entire Cap (opti-eCap) and one optimized Cap deleted nuclear location signal (NLS) (opti-dCap) gene fragments encoding the same amino acid sequence with wt-eCap were synthesized based on the codon bias of E. coli. Those gene fragments were inserted into the pET30a expression vector. One recombinant strain with the highest expressed soluble eCap from four entire Cap (one wt-eCap and three opti-eCap) and one recombinant strain expressed opti-dCap were selected for further purification. The purified eCap and dCap were identified by transmission electron microscopy (TEM), a large number of round hollow particles with a diameter of 10 nm virus-like particles (VLPs) were observed in eCap, whereas irregular aggregation of proteins observed in dCap. After formation the VLPs were applied as a coating antigen to establish an indirect ELISA (I-ELISA) for detection of PCV3-specific antibody in swine serum. 373 clinical swine serum samples from China collected in 2019 were tested utilizing the VLP-based I-ELISA method under optimized conditions. To the best of our knowledge, this is the first report of self-assembly into VLPs of PCV3 recombinant Cap. Our results demonstrated that the VLP-based I-ELISA will be a valuable tool for detecting the presence of PCV3 antibodies in serum samples and will facilitate screening of large numbers of swine serum for clinical purposes.

Pathogenic Characterization of a Porcine Circovirus Type 3 Isolate from Heilongjiang, China

“…To further characterize the organ lesions induced by PCV3 DB-1 infection, the lungs and lymph nodes were examined on a microscopic level for histopathological damage. H&E staining of PCV3 DB-1-infected piglets showed lymphocyte reduction, companying by inflammatory cell infiltration in the lymph nodes (Figure 3(a)), as well as epithelial cell proliferation, inflammatory cells infiltration, and thickened alveolar septum in the lungs (Figure 3 To better understand the humoral immune response elicited by PCV3 DB-1 challenge, the anti-PCV3 antibody titer in the serum samples was assessed by an indirect ELISA based on PCV3 recombinant rCap-VLPs [23]. Throughout the experiment, the IgG antibody response varied among the individuals (Figure 5).…”

“…To better understand the humoral immune response elicited by PCV3 DB-1 challenge, the anti-PCV3 antibody titer in the serum samples was assessed by an indirect ELISA based on PCV3 recombinant rCap-VLPs [ 23 ]. Throughout the experiment, the IgG antibody response varied among the individuals ( Figure 5 ).…”

“…In our study, 5 of nine PCV3 DB-1 inoculated piglets showed an obvious trend in the IgG antibody response. The indirect ELISA method used in our study based on PCV3 recombinant rCap-VLPs [ 23 , 30 ] might conserve the conformational epitopes and had the ability to detect IgG antibodies against the conformational epitope of the PCV3. Antibodies against rCap-VLPs of PCV3 that appeared in piglets with a specific immune status may be inflammatory factors that alert immune cells to respond to the viral infection; however, whether these antibodies can neutralize PCV3 requires further investigation.…”

“…Serology. Serum antibodies against PCV3 were detected using a modified indirect enzyme-linked immunosorbent assay (ELISA) based on PCV3 recombinant virus-like particles (VLPs) [23]. The optical density (OD) was read at 450 nm using an ELISA plate reader (PE, USA).…”

Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV

“…Mammalian cells are increasingly being used to generate VLP-based vaccines as exemplified by porcine circovirus (PCV), porcine parvovirus (PPV), Lassa virus (LASV), Marburg virus (MARV), and Ebola virus (EBOV) VLPs [ 35 ]. Due to the ability of VLPs to trigger strong immune response and to induce antibody production, VLPs are considered to be a potential novel vaccine candidate [ 24 , 36 ].…”