E coli bl21 de3 competent cell

Taq DNA polymerase, restriction endonucleases, and PCR reagents were obtained from Takara (Beijing, China). In-Fusion HD Cloning Kit (Takara Beijing, China), plasmid pEASY-E2, and E. coli BL21(DE3) competent cells (TransGen, Beijing, China) were adopted in the cloning process. The purification was performed with Ni-NTA Agarose resin (Qiagen, Hilden, Germany). p-Nitrophenyl-α-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-galactopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-mannopyranoside, and 2-nitrophenyl-β-D-glucopyranoside were supplied by Shanghai Yuanye Bio-Technology (China). The vector pCC1FOS and E. coli EPI300 were purchased from Epicentre. The remaining chemicals reagents were of analytical grade except for those specified. The cultivation environment of the strains E. coli is at 37°C in the Luria–Bertani (LB) liquid medium, in which the salt concentrations can reach their final stages, encompassing the salt showed in the normal LB medium. In order to select certain plasmids, 12.5 μg/ml chloramphenicol (Cm) or ampicillin (100 μg/ml) was used as an alternative of the medium. Previously constructed fecal microbial metagenomic libraries of N. pygmaeus and B. frontalis were used (Xu et al., 2014 (link); Dong et al., 2016 (link)).

The ASFV P72 gene sequence was from strain ASFV Georgia 2007/1 (GenBank: FR682468.2), and the prokaryotic expression recombinant plasmid pET-30a-p72 was synthesized and constructed by Wuhan Jinkairui Biology Co, Ltd. The recombinant plasmid pET-30a-p72 was transformed into E. coli BL21 (DE3) competent cells (TransGen Biotech, China) and inoculated in Luria–Bertani (LB) liquid medium containing kanamycin (100 μg/mL) and cultured at 37 ℃ and 220 r/min. When the OD_600nm value was 0.6–0.8, IPTG (final concentration was 1 mmol/L) was added to induce the expression of 6-h bombardment. Purified rP72 protein was analyzed by SDS-PAGE electrophoresis after purification by Ni Sepharose. The antigenicity of ASFV was identified by western blot method with ASFV inactivated positive serum (1:200) as the first antibody and goat anti-porcine IgG-HRP (1:10,000) as the second antibody.

The previously constructed recombinant plasmids were transformed into E. coli BL21(DE3) competent cells (TransGen) by heat shock at 42°C for 30 s and screened with kanamycin (50 μg/ml). Single colonies were selected from the plate and inoculated into LB medium with kanamycin (50 μg/ml) at 37°C. When the OD600 was 0.6, isopropyl-b-D-thiogalactoside (0.2 mM) was added to the culture medium and further incubated for 6 h at 37°C. The cells were centrifuged for 20 min at 8,000 ×g to collect the bacteria. Sonication was performed, and the supernatant was separated and precipitated by centrifugation for 20 min at 8,000 ×g. The precipitate was resuspended in Tris-HCl (20 mmol/l, pH 8.0) buffer containing 8 M urea at a ratio of 1:10 (w/v). The mixture was incubated at 4°C overnight and then filtered through a 0.45 μm membrane. The denatured p170 and p170-RBD proteins were then further purified with Ni-affinity chromatography (GE Healthcare, USA) using the ÄKTA explore system (GE Healthcare). The column was equilibrated with Tris-HCl buffer (20 mmol/l, pH 8.0) containing 8 M urea. The concentration of imidazole was increased gradually, and the corresponding peak was collected. Purified protein was refolded by reducing the urea concentration gradually with a dialysis bag, followed by concentration with 10 kDa and 100 kDa ultrafiltration devices successively and then stored at -80°C.

Plasmids of the positive clones were isolated from the phage-ELISA and double digested using the restriction enzymes NcoI and NotI. The digested VHH fragments were ligated into the pET-25b ( +) –SBP plasmid and digested using the same restriction endonuclease with T4 DNA ligase. The pET-25b ( +) vector carried a streptavidin-binding protein that was constructed and preserved by the Public Health Department of the School of Veterinary Medicine, Inner Mongolia Agricultural University. The products were transformed into E. coli BL21 (DE3) competent cells (TransGen Biotech, Beijing, China). The expression of recombinant sdAb was induced using isopropyl-β-d-thiogalactoside (IPTG) (Solarbio Life Sciences, Beijing, China) for 8–12 h. The cells were collected and sonicated. The precipitate and supernatant were collected and analyzed using SDS-PAGE. Ni–NTA Sefinose™ Resin (Sangon Biotech, Shanghai, China) was used to purify the expressed recombinant single domain antibodies, and the purified sdAbs were analyzed using SDS-PAGE.

After amplification, the coding region of MaCD68 was digested and inserted into the pET32a vector to construct a recombinant plasmid that was transformed into E. coli BL21 (DE3)-competent cells (TransGen Biotech, Beijing, China) using the primers listed in Table 1. The recombinant MaCD68 (rMaCD68) protein induced by IPTG at a final concentration of 0.5 mM was purified using a Ni-Agarose His-tagged Protein Purification Kit. The purified rMaCD68 protein was verified by mass spectrometry analysis and the concentration was detected using a BCA Protein Assay Kit (CoWin Biosciences, Taizhou, Jiangsu, China).
Anti-MaCD68 polyclonal antibody was prepared by subcutaneously immunizing (thrice) two Japanese white rabbits with 500 μg of recombinant MaCD68. The first injection was a mixture of recombinant protein and complete Freund’s adjuvant (Bio Basic, Inc., Toronto, ON, Canada). After an interval of 2 weeks, secondary immunizations were performed with rMaCD68 mixed with incomplete Freund’s adjuvant. Antisera were collected 10 days after the last immunization. Additionally, the antiserum was purified using a protein A/G assay, and the specificity of the antibody was tested by Western blotting (see below). Normal rabbit serum collected before the primary injection was used as a negative control.

F81 cells, vesicular stomatitis virus with green fluorescent protein (VSV-GFP), and mink parvovirus virulent strain SMPV-11 were cultured in our laboratory. E. coli BL21 (DE3) competent cells were purchased from TransGen Biotech Co., LTD (Beijing, China). The expression vector pPICZαA and P. pastoris strain X33 were purchased from Invitrogen. The culture supernatant was concentrated using an ultrafiltration system with a molecular weight (MW) cutoff of 5 kDa (Vivaflow 200 Hydrosart; Sartorius, Goettingen, Germany). X33 transformants were maintained in YPD medium (1% yeast extract, 2% tryptone, 2% glucose) containing 30% (v/v) glycerol and were stored at −80°C.
Six male rats (200 g body weight) were purchased from a fur farm in Jilin Province, China. This study was approved by the Institute of Special Economic Animal and Plant Sciences of the Chinese Academy of Agricultural Sciences (CAAS; No. ISAPSAEC-2021-27M), and all sampling procedures complied with the guidelines of the Institutional Animal Care and Use (IACUS) regarding the care and use of animals for scientific purposes. This study was also approved by the Laboratory Animal Management and Welfare Ethics Committee of the Institute of Special Economic Animal and Plant Sciences.