Virus and cells. PK-15 cells were purchased from ATCC (CCL-33), and propagated in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum, 100 ug/mL streptomycin, and 100 IU/mL penicillin (GIBCO, Invitrogen). The attenuated strain, NADL-2, of PPV was obtained from the National Institute of Veterinary Drug Control.
Gene synthesis and plasmid construction. According to the advanced structure and GC content of the PPV VP2 gene sequence (JQ710896.1) and the codon usage frequency of the Escherichia coli host, the VP2 optimized gene sequence (amino acids 156–438) was optimized using software of Rare Codon Caltor and synthesized by Wuhan Jinkaili Bioengineering Co., Ltd. Hind III and BamH I restriction sites were introduced at the 5' and 3' ends of the VP2 optimized gene (amino acids 156–438), respectively. The pCold vector and the VP2 optimized gene fragment (amino acids 156–438) were double digested and ligated with T4 ligase. The ligation product was transformed into the E.coli DH5α competent state and cultured at 37 °C for 12 h. The selected positive clones were cultured in IR800 medium for 12 h. Plasmids were extracted and sequenced. The correct plasmid was identified as pCold-VP2.
Expression and purification of PPV VP2 protein. Rosetta2 (DE3) cells (Novagen, Shanghai, China) transformed with pCold-VP2 were cultured in LB medium supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, were grown at 37 ℃ until achieving the logarithmic phase (at OD600 of 0.6), and induced by Isopropyl-thio-galactoside (IPTG) at a final concentration of 0.1 mM for 16 h at 18 ℃. The re-suspended cells were lysed by sonication on ice for 8 × 4 s with 3 s intervals. The lysate was centrifuged at 10,000 g for 15 min at 4 ℃, and the supernatant and the pellet resuspended in PBS were both analyzed by SDS–PAGE to observe the solubility of the target protein. The fusion proteins in the inclusion bodies were denatured using 8 μM urea at 4 ℃ overnight, and the supernatant was retained after centrifugation. His-tagged VP2 fusion proteins were bound to Ni-NTA resin (Qiagen, Hilden, Germany) and purified following the manufacturer’s instructions. Finally, freshly purified proteins were treated with Detoxi-GelTM Endotoxin Removing Gel (Thermo Fisher Scientific, Waltham, USA) to remove endotoxin derived from bacterial culture in the fusion proteins. Aliquots of purified protein were stored at –80 ℃.
Production of mouse anti-VP2 polyclonal antibody. One group of Balb/c mice （Qinglongshan Animal Breeding Farm，Nanjing, China）were immunized at 100 μg VP2 protein with Freund's complete adjuvant (1:1, Sigma-Aldrich, USA) to form an emulsion. Another group infected with the same dose of sterile saline was set as negative control. After the first immunization, the second and third immunizations were performed after Freund's incomplete adjuvant (1:1) emulsification every two weeks. After the third immunization, serum was collected from the orbital blood and stored in a refrigerator at -80 ° C, similarly as measured in previous study. The mice were sacrificed by cervical dislocation without anesthesia. After confirming the death, the mice were put into a freezer for centralized processing.
Indirect immunofluorescence (IFA). PK-15 cells were infected with PPV for 48 hours and fixed with 4% paraformaldehyde, permeabilized with 0.1% X-Triton, and blocked with 10% sheep serum. PK-15 cells were incubated with mouse anti-VP2 polyclonal antibody (1:500) for 2 h at 37 ℃, washed with PBST 3 times, incubated with goat anti-mouse antibody (Alexa Fluor＆488, abcam, USA) for 1 hour at 37 ℃, and washed with PBST 3 times. The nucleus was dyed with DAPI for 20 minutes and observed with a confocal microscope.
Antibody labeled. The standard PPV positive antibody (National Institute of Veterinary Drug Control) was conjugated to Cyanine3 fluorescent dye using a Lightning-Link® HRP conjugation kit (Innova Biosciences, UK).
Protein chip preparation. Before printing, 50% of the moisture of the spotter was prepared at 4 ℃. VP2 protein of 0–0.4 mg/mL was printed on epoxy-coated glass slides with 2 × printing buffer (Capital Biochip Biotechnology Co. Ltd, Beijing, China); each dilution of VP2 protein was printed 3 times using the SmartArrayer™ microarray spotter (Capital Biochip Biotechnology Co. Ltd, Beijing, China). The spotted chip was placed in a 37 ℃ molecular hybridization instrument and hydrated for 10 h. After 5 min washing with PBST, 1% BSA was added to each sub-array for blocking. Then the prepared slides could be used immediately or could be stored at 4 ℃ after centrifuging and drying. The diluted standard PPV positive antibody serum was added to the chips and incubated for 1 h at 37 ℃. After washing and drying, the protein chip was observed by scanning with a Luxscan-10K/A chip scanner (Capital Biochip Biotechnology Co. Ltd, Beijing, China).
Establishment of visible protein chip. To determine the optimal concentration of diagnostic antigen, PPV VP2 protein were diluted to 0.002 mg/mL, 0.02 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, and 0.4 mg/mL with 2 × printing buffer, each concentration was diluted 3 times. The blocked chip was added with 100-fold diluted positive control serum and incubated at 37 ℃ for 1 h. After washing with PBST three times, 100-fold diluted rabbit anti-pig IgG was added on the chips and incubated at 37 ℃ for 1 h. After washing with PBST three times, the well-mixed silver staining solution A and B (Sigma-Aldrich, St. Louis, USA) were added into each well, and then the results were observed directly. Once the antigen concentration was determined, the optimized concentration of VP2 protein was printed on epoxy-coated glass slides for subsequent experiments. The positive control serum was diluted to 10-, 100-, 1000-, 2000-, 4000-, 5000-, and 6000-fold to determine the optimal serum dilution. The incubation time of primary and secondary antibodies was set to 30 min, 45 min, and 1 h, respectively, and a total of 9 groups of time gradient tests were performed to determine the optimal antigen–antibody reaction time.
Establishment of Cy3-labeled protein chip. To determine the optimal antigen concentration, PPV VP2 proteins were diluted with a series of concentrations (0.002, 0.02, 0.05, 0.1, and 0.2 mg/mL) with 2 × printing buffer, each concentration was diluted 3 times. The blocked chip was added with 100-fold diluted positive porcine serum and incubated at 37 °C for 1 h. After washing with PBST, 100-fold diluted Cy3-labelled rabbit anti-porcine IgG was added and incubated at 37 ℃ for 1 h. After washing with PBST for 5 min, the chip was centrifuged and dried. The results were observed by scanning with a Luxscan-10K/A chip scanner. Once the antigen concentration was determined, the optimized concentration of VP2 protein was printed on epoxy-coated glass slides for subsequent experiments. PPV antibody positive serum was diluted 10–25,600-fold to determine the optimal serum dilution ratio. The incubation time of primary and secondary antibodies was set to 30 min, 45 min, and 1 h, respectively, and a total of 9 groups of time gradient tests were performed to determine the optimal antigen–antibody reaction time.
Determining the positive–negative threshold and specificity.
Thirty known negative sera against PPV were added to the prepared protein chips to determine the cut-off value for antibody detection. In addition, the protein chip was divided into four microarrays, and three spots were repeated in each array. CSFV, PPV, JEV, and PRRSV positive serum were added to the corresponding arrays to verify the specificity.
Detection of clinic samples. The established PPV visible protein chip was used to detect 9 negative and 42 positive swine serum samples that had been detected using an indirect ELISA assay (Wuhan Keqian Biology Co., Ltd, China). The serum dilution ratio of the ELISA is 2-fold. The established PPV Cy3-labeled protein chip detection microarray was used to detect 15 negative and 19 positive swine serum samples that had been detected by indirect ELISA. Then, 120 clinical swine serum samples（farmer permission） from the pig farms in Jiangsu province were tested using the two established protein chip technologies and compared with commercial PPV ELISA antibody detection kits.