Ni affinity chromatography

The correctly constructed expression plasmids pET28a/TsDNase II-7 were transformed into Escherichia coli BL21 (DE3) (Novagen, USA) for expression. The positive clone verified by DNA sequencing was inoculated in 10 mL of LB medium with Kanamycin (100 μg/mL) and incubated at 225 rpm at 37 °C overnight. Then 1 L of LB medium supplemented with the same antibiotics were mixed with 10 mL of the overnight culture. Temperature at 37 °C with 225 rpm until an OD of 0.5–0.6 was achieved at 600 nm. After induction of the expression with 1 mM isopro-pyl-β-D-thioga-lactopyranoside (IPTG) at 37°C for 6 h, bacterial precipitate was harvested after 30 min ultrasonic crushing on ice using centrifugation at 3,000 g for 25 min and subjected to protein purification. The rTsDNase II-7 was purified by Ni-affinity chromatography (Qiagen, California) according to the manufacturer’s instructions.
A rabbit was subcutaneously immunized with approximately 500 μg of purified rTsDNase II-7 (1 mg/mL) emulsified with Freund’s complete adjuvant (FCA; Sigma, USA) and boosted four times at a two-week interval. Fourteen days after the final immunization, blood samples were taken from immunized rabbit heart and a polyclonal antibody against rTsDNase II-7 at a titer of 1:256,000 was analyzed by ELISA. Affinity-purified antibodies were used for subsequent Western blot and immunofluorescence assays (IFAs).

According to the complete genome sequence of M. bovis strain PG45 (23 (link)). Nine TGA codons exist in the coding gene sequence of MBOVPG45_0375. TGA is a universal termination codon but it encodes tryptophan in mycoplasmas. When cloning a mycoplasma gene in the E. coli expression system, the presence of TGA codon can lead to the early termination of gene translation. The intact open reading frame of the gene sequence of MBOVPG45_0375, stop codon-included and TGA-corrected, was synthesized by Sangon Biotech (Shanghai, China). Expression was performed as previously described (24 (link)). Briefly, the gene was digested (BamH I and Xho I) and ligated into the pET-28a plasmid to produce pET-MBOVPG45_0375. The plasmid was transformed into E. coli BL21(DE3) competent cells. Escherichia coli BL21(DE3)/pET-28a-MBOVPG45_0375 transformants were cultivated for 6 h at 37°C with constant shaking (200 r/min), followed by induction using 1 mM isopropyl-β-D-thiogalactoside (IPTG) for 8 h. Cells were collected and disrupted by sonication. The supernatant was separated by centrifugation and the precipitate was re-suspended in phosphate buffer saline (PBS, 10 mM, PH 7.2). Purification was performed using Ni-Affinity Chromatography (Qiagen, NY, USA) following previously described protocols (24 (link)), and the solubility and purity of r0375 were analyzed by SDS-PAGE.

T. spiralis (ISS533) was maintained in female ICR mice. Muscle larvae (ML) were recovered from infected mice using a modified pepsin-hydrochloric acid digestion method as previously described [20 (link)]. Adult worms were collected from the intestines of infected mice four days following oral larval challenge. Crude adult worm antigens were prepared from homogenized worm extracts based on a previously described protocol [18 ]. The anti-Ts-Pmy monoclonal antibody (mAb) 9G3 that specifically recognized Ts-Pmy was previously produced [19 (link)]. Recombinant Ts-Pmy (rTs-Pmy) with a His-tag at the C-terminus was expressed in baculovirus/insect cells (Invitrogen, USA) and purified by Ni-affinity chromatography (Qiagen, USA). Ts87 (38 kDa) was an excretory-secretory antigen of T. spiralis identified previously [21 (link)]. In this study, recombinant Ts87 (rTs87) was used as a non-relevant protein control.

The lysA nucleotide sequences were amplified from Halomonas sp. strain A40-4, Thalassospira sp. strain A40-3, Alteromonas sp. strain B31-7, and Thalassospira sp. strain B30-1 by the PCR program shown in Table S3 with FastPfu DNA polymerase (Trans, China) and the primers shown in Table S4 and then introduced into a pET22b expression plasmid with an In-Fusion HD cloning kit (TaKaRa, Japan). The constructed recombinant plasmids were transformed into E. coli BL21(DE3). Transformants were cultured at 37°C and 180 rpm in LB liquid medium containing 100 mg mL⁻¹ ampicillin. When the OD₆₀₀ of the cultures reached approximately 0.6, 1 mM isopropyl-b-d-thiogalactopyranoside (IPTG) was added to the culture for the induction of protein expression. Then, the cultures were incubated at 15°C and 110 rpm for 14 h. After incubation, the cells in the cultures were harvested, resuspended in lysis buffer (50 mM Tris-HCl, 100 mM NaCl; pH 8.0), and disrupted by high pressure using a JN-02C low-temperature ultrahigh-pressure continuous-flow cell disrupter (JNBIO, China). The recombinant His-tagged LysA proteins in the resulting extracts were purified by Ni affinity chromatography (Qiagen, USA) and desalted with disposable PD-10 desalting columns (GE Healthcare, Sweden). Protein concentrations were determined by using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, USA).

The gene H8 encoding a lipolytic enzyme was amplified from the fosmid DNA using the primer pair of H8_F (5′-GGGAATTCCATATGCAGTCTGGCACGGTGAG-3′, NdeI digestion site was underlined) and H8_R (5′-CCGCTCGAGCGCCACCGCCGGTTGCGCC-3′, XhoI digestion site was underlined), and cloned into the expression vector pET-22b.
The constructed plasmid pET-22b-H8 was transformed into E. coli BL21 (DE3). Transformants were cultured at 37°C and 180 rpm in LB liquid medium containing 100 μg/mL ampicillin. When the OD₆₀₀ of cells reached approximately 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for the induction of protein expression. Then, the culture was incubated at 20°C and 110 rpm for 20 h. After incubation, the cells in the culture were harvested, resuspended in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0) and disrupted by pressure. The recombinant His-tagged protein in the extract was first purified by Ni affinity chromatography (Qiagen, USA), and further purified by gel filtration chromatography on a Superdex 200 column (GE healthcare, Sweden). Protein concentrations were determined by using the Pierce BCA Protein Assay Kit (Thermo Scientific, USA).

DNA encoding TsPmy without signal peptide was cloned into the expression vector pET-28a (+). The recombinant TsPmy protein (rTsPmy) was expressed and identified in an Escherichia coli (BL21) expression system in our laboratory. The rTsPmy with a His-tag at the C terminus was expressed in E. coli under 1 mM IPTG induction and purified with Ni-affinity chromatography (Qiagen, United States) as previously described (Yang et al., 2008 (link)).