Protein purification kit

The truncated cap gene (121–813 nt, deleted 120 nt at the 5′ terminal) was expressed as a His-tagged protein in E. coli with the expression vector pET-32a (Novagen). Canine circovirus strain XF16 obtained previously by our team (GenBank accession No. MF797786) was used as template. The gene was amplified through PCR with the forward primer 5′-UGGATCCLINECTGACAGCTGATTG−3′ and the reverse primer 5′-UCTCGAGLINETTACAACTGGCG−3′. The PCR product was digested with BamHI and XhoI, and cloned into pET-32a vector. The resulting recombinant plasmid pET-32a-cap was sequenced by Comate Biotech., Changchun, China and transformed into E. coli Rosetta cells. The recombinant E. coli Rosetta cells were induced to express his-tagged recombinant protein and purified using protein purification kit (Qiagen, Hilden, German) according to the manufacturer's instructions. rCap was identified through Western blot by utilizing HRP-conjugated mouse anti-His MAb (Sigma, Missouri, USA) as antibodies.
To evaluate the potential cross-reactivity of the rCap protein in Western blot, we used the positive canine serum samples of CAV2, CPV2, RV, and CDV. Sera from the laboratory animal center and those from fetus dog obtained by cesarean section were used as negative controls. These samples were assayed in duplicate.

The Cas expression plasmids were individually transformed into E. coli BL21 (DE3) and expression of the His-tagged Cas proteins was performed following the instruction of the protein purification kit (Qiagen, Valencia, CA, USA). Single colonies of transformed cells were cultivated overnight, followed by 1/100 dilution into 100 ml of LB media containing 100 µg ml⁻¹ ampicillin. The cells were firstly incubated at 37°C to an OD₆₀₀ of 0.6–0.8, then transferred to a shaker and induced with isopropyl β-D-1-thiogalactopyranoside in a final concentration of 0.5 mM at 16°C. After continuously shaking for 22 h, cells were harvested, lysed and purified using Ni-NTA resin (Qiagen, Valencia, CA, USA). The purified proteins were desalted with desalting column (GE Healthcare, Chicago, IL, USA) using AKTA system (GE Healthcare, Chicago, IL, USA), and finally confirmed by SDS-PAGE electrophoresis. Throughout purification, we used a buffer containing 20 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM DTT and 2 mM EDTA for lysis, washing and elution.