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Construction of engineered E. coli strains and protein purification. (A) PCR identification of positive E. coli clones. Lanes 1–3 correspond to BL21-pET28-ST-PCV2 Cap, BL21-pET28-ST-PCV3 Cap, and BL21-pET28-SC-mi3, respectively. (B–D) Expression and purification of recombinant proteins ST-PCV2 Cap, ST-PCV3 Cap, and SC-mi3, respectively. Lanes 1–6 represent whole bacterial lysate, pellet after sonication, supernatant after sonication, Ni column flow-through, wash fraction, and purified protein, respectively. (E) Western blot validation of antigen proteins. Lanes 1, 2 correspond to ST-PCV2 Cap and ST-PCV3 Cap, respectively. (F) Endotoxin removal from recombinant protein solutions. Color intensity represents endotoxin content; numbers indicate endotoxin content (EU/mL).
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Construction of engineered E. coli strains and protein purification. (A) PCR identification of positive E. coli clones. Lanes 1–3 correspond to BL21-pET28-ST-PCV2 Cap, BL21-pET28-ST-PCV3 Cap, and BL21-pET28-SC-mi3, respectively. (B–D) Expression and purification of recombinant proteins ST-PCV2 Cap, ST-PCV3 Cap, and SC-mi3, respectively. Lanes 1–6 represent whole bacterial lysate, pellet after sonication, supernatant after sonication, Ni column flow-through, wash fraction, and purified protein, respectively. (E) Western blot validation of antigen proteins. Lanes 1, 2 correspond to ST-PCV2 Cap and ST-PCV3 Cap, respectively. (F) Endotoxin removal from recombinant protein solutions. Color intensity represents endotoxin content; numbers indicate endotoxin content (EU/mL).
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Construction of engineered E. coli strains and protein purification. (A) PCR identification of positive E. coli clones. Lanes 1–3 correspond to BL21-pET28-ST-PCV2 Cap, BL21-pET28-ST-PCV3 Cap, and BL21-pET28-SC-mi3, respectively. (B–D) Expression and purification of recombinant proteins ST-PCV2 Cap, ST-PCV3 Cap, and SC-mi3, respectively. Lanes 1–6 represent whole bacterial lysate, pellet after sonication, supernatant after sonication, Ni column flow-through, wash fraction, and purified protein, respectively. (E) Western blot validation of antigen proteins. Lanes 1, 2 correspond to ST-PCV2 Cap and ST-PCV3 Cap, respectively. (F) Endotoxin removal from recombinant protein solutions. Color intensity represents endotoxin content; numbers indicate endotoxin content (EU/mL).
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e coli bl21 de3 plyss cells  (Thermo Fisher)
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Construction of engineered E. coli strains and protein purification. (A) PCR identification of positive E. coli clones. Lanes 1–3 correspond to BL21-pET28-ST-PCV2 Cap, BL21-pET28-ST-PCV3 Cap, and BL21-pET28-SC-mi3, respectively. (B–D) Expression and purification of recombinant proteins ST-PCV2 Cap, ST-PCV3 Cap, and SC-mi3, respectively. Lanes 1–6 represent whole bacterial lysate, pellet after sonication, supernatant after sonication, Ni column flow-through, wash fraction, and purified protein, respectively. (E) Western blot validation of antigen proteins. Lanes 1, 2 correspond to ST-PCV2 Cap and ST-PCV3 Cap, respectively. (F) Endotoxin removal from recombinant protein solutions. Color intensity represents endotoxin content; numbers indicate endotoxin content (EU/mL).
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Construction of engineered E. coli strains and protein purification. (A) PCR identification of positive E. coli clones. Lanes 1–3 correspond to BL21-pET28-ST-PCV2 Cap, BL21-pET28-ST-PCV3 Cap, and BL21-pET28-SC-mi3, respectively. (B–D) Expression and purification of recombinant proteins ST-PCV2 Cap, ST-PCV3 Cap, and SC-mi3, respectively. Lanes 1–6 represent whole bacterial lysate, pellet after sonication, supernatant after sonication, Ni column flow-through, wash fraction, and purified protein, respectively. (E) Western blot validation of antigen proteins. Lanes 1, 2 correspond to ST-PCV2 Cap and ST-PCV3 Cap, respectively. (F) Endotoxin removal from recombinant protein solutions. Color intensity represents endotoxin content; numbers indicate endotoxin content (EU/mL).

Journal: Frontiers in Veterinary Science

Article Title: Construction and immunogenicity evaluation of a bivalent nanoparticle based on mi3 displaying porcine circovirus type 2 and type 3 capsid proteins

doi: 10.3389/fvets.2026.1862938

Figure Lengend Snippet: Construction of engineered E. coli strains and protein purification. (A) PCR identification of positive E. coli clones. Lanes 1–3 correspond to BL21-pET28-ST-PCV2 Cap, BL21-pET28-ST-PCV3 Cap, and BL21-pET28-SC-mi3, respectively. (B–D) Expression and purification of recombinant proteins ST-PCV2 Cap, ST-PCV3 Cap, and SC-mi3, respectively. Lanes 1–6 represent whole bacterial lysate, pellet after sonication, supernatant after sonication, Ni column flow-through, wash fraction, and purified protein, respectively. (E) Western blot validation of antigen proteins. Lanes 1, 2 correspond to ST-PCV2 Cap and ST-PCV3 Cap, respectively. (F) Endotoxin removal from recombinant protein solutions. Color intensity represents endotoxin content; numbers indicate endotoxin content (EU/mL).

Article Snippet: The pET28a(+) plasmid for recombinant protein expression and BL21(DE3) E. coli competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.

Techniques: Protein Purification, Clone Assay, Expressing, Purification, Recombinant, Sonication, Western Blot, Biomarker Discovery