Aqueous two-phase system (ATPS) has great potential in industrial applications of bio-separations and bio-reactions. However, its large-scale application is limited by recovery difficulty of phase systems. In this paper, a recyclable ATPS was prepared by two pH-responsive copolymers (P ADB4.99 and P MDM7.08 ) and applied for purification of porcine circovirus type 2 Cap protein fermentation broth (PCV2 Cap protein). Phase separation mechanism was studied by using low-field nuclear magnetic resonance (LF-NMR). The results showed that relatively desirable ATPS were formed successfully when two copolymer concentrations were 4%–6% (w/w) with pH 7.5–8.6. The main parameters, such as copolymer concentration, temperature, pH, type and concentration of salts, were investigated. The results demonstrated that the best ATPS consisted of 4% (w/w) P ADB4.99 and 6% (w/w) P MDM7.08 , the optimal partition coefficient (K) and extraction recovery (ER) of PCV2 cap protein were 0.25 and 94.2% in the presence of 50 mM Li 2 SO 4 , 3.53 and 93.4% in the presence of 40 mM KCl, respectively, at 30 °C, pH 8.1. The maximum purity of PCV2 Cap protein was 88.4%. In addition, the two phase-forming copolymers could be recycled by adjusting solution pH to their isoelectric points (pI), with recoveries of over 97.5%.
The vaccine against porcine circovirus type II (PCV2) is mainly composed of virus capsid proteins. An efficient separation method of PCV2 Cap protein has not been developed. Affinity precipitation methods with high selectivity and recoverability based on the thermo-responsive affinity polymer (P NDBN -Ni) were introduced to separate the PCV2 Cap protein. When the temperature was above the polymer LCST (36.1 °C), P NDBN -Ni could be precipitated with a recovery of above 95.00%. The affinity ligand immobilized on the polymer could specifically bind the proteins. The optimal conditions for affinity precipitation were a pH of 6.0 with a ligand density of 56.22 μmol/g polymer at 25.0 °C. The final results were detected by electrophoresis. The interactions between proteins and ligands were determined by the change in ligand state as seen using X-ray photoelectron spectra (XPS).
Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion.Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus.Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells.Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.
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