2.1 Reagents

The reagents used in this study, 2×EasyTaq® PCR SuperMix (+dye) (AS111), 2×TransStart® FastPfu Fly PCR SuperMix (-dye) (AS231), Trans DNA Marker II (BM101), Trans8K DNA Marker (BM151), FlyCut® EcoRI (JE201), FlyCut® NcoI (JN101), EasyPure® Quick Gel Extraction Kit (EG101), T4 DNA Ligase (FL101), EasyPure® Plasmid MiniPrep Kit (EM101), Agarose, GelStain, BL21 (DE3) Chemically Competent Cell (CD601) and ProteinRuler® II (12–120 kDa) (DR201) were all purchased from TransGen Biotech.

2.2 Sequences and plasmids

The fusion genes were designed based on the sequences of PRV gB, gC, and gD genes. The gB aa 59–126, gC aa 156–290, and gD aa 78–183 were selected for reorganization. According to the properties of amino acids, 2–4 glycines were inserted between the epitopes, and then the epitopes were connected in series on the ORF without signal peptide. Sequences of PRV-gB/C, gC/D, and gB/C/D were all synthesized by Biosune Biotechnology Co., Ltd. The recombinant plasmids were validated using sequencing in Sangon Biotech. The synthesized sequences were inserted into the pET-28a⁺ vector (between NcoI and EcoRI) to construct the recombinant plasmids.

2.3 Polymerase chain reaction (PCR) and enzyme digestion identification

PCR was preformed following the instructions. Details of the PCR kit were in the reagents section. The primers used in this study were synthesized from Biosune Biotechnology Co., Ltd., including gB/C (F-CCCAATCCATGGCCGCAGCAGTTACCCGTGCAG and R-CCCAATGAATTCGGAAACGCGGCATCAACCGGA), gC/D(F-CCCAATCCATGGCCTATTTTGATGAACCGCCGCGT and R-CCCAATGAATTCGGCAGGGCAACCATAAAATCTGTCAG) and gB/C/D (F-GCAGCAGTTACCCGTGCAGCAA and R-CAGGGCAACCATAAAATCTGTCAG).

The extracted recombinant plasmids were verified by double enzyme digestion according to the instructions. After added 1 μl/50 μl NcoI and EcoRI, plasmids were incubated at 37°C for 15 min. Double-enzyme digestion products were detected by agarose gel electrophoresis of 1.5%.

2.4 Induction of protein expression

Three verified strains were seeded in 5 ml LB/Kan⁺ medium and cultured at 37°C with 250 rpm. When OD600 reached 0.5, 1 mM IPTG was added to induce protein expression. After the induction for 2, 4, and 6 h, the bacterial solution was collected and then boiled in a protein loading buffer at 95°C for 5–10 min.

2.5 SDS-PAGE and western blot

The protein was separated with 5% concentrated gel and 12% separated gel. Thirty micrograms of protein samples were loaded to the lane and electrophoresed at 70 V for 30 min and then 90 V until the indicator reached at about 0.5 cm from the lower end of the gel. The samples were stained with Coomassie brilliant blue at room temperature for 1 h. After decolorization, photos were taken.

For western blot analysis, the proteins were transferred from sodium dodecyl sulfate polyAcrylamide gel electrophoresis to polyvinylidene fluoride (PVDF) membranes at 200 mA for 90 min. After blocking with 5% bachelor of science in agriculture at room temperature for 2 h, PVDF membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 2 h. The proteins were maintained with chemiluminescence reagent for 2 min and then were exposed for photography. Details of antibodies were shown in the reagents section.

2.6 Establishment of indirect ELISA for recombinant protein

According to matrix titration, different concentrations of recombinant proteins (0.5, 1, 3, and 5 mg/L) and serums with different dilutions (1:20, 1:50, 1:100, 1:200) were added to 96-well plates and incubated at 37°C for 60 min. Recombinant proteins were incubated with Enzyme labeled antibody at 37°C for 60 min. Then, 100 μl of TMB substrate chromogenic solution was added and maintained at 37°C for 10 min. OD650 value was measured by a microplate reader.

3.1 Construction of recombinant expression vectors

First, the gB/C, gC/D, and gB/C/D fusion sequences were designed and synthesized according to the sequence information in NCBI. Then, the target sequences were cut off and connected to the prokaryotic expression vector pET-32a. The target bands were successfully amplified by PCR assay (Figure 1A).

The accuracy of the synthesized sequence was identified by enzyme digestion and sequencing. As shown in Figure 1B, after double enzyme digestion, the fragment lengths of gB/C, gC/D, and gB/C/D were consistent with the expected result. According to the sequencing results shown in Supporting Information: Figure, the bases of the BC and CD fragments were completely consistent with those of wild-type fragments, and the bases of the gB/C/D fragment and the wild-type fragment are more than 99% identical.

3.2 Expression of recombinant proteins

The identified recombinant plasmids were transformed into BL21 Escherichia coli competent cells to express the recombinant proteins. IPTG was used to induce protein expression for 0, 2, 4, and 6 h. According to the software calculation, the molecular weights of gB/C, gC/D, and gB/C/D recombinant proteins were 21.51, 27.17, and 33.92 kDa, respectively. As shown in Figure 2A, the molecular weight of recombinant proteins gB/C, gC/D, and gB/C/D were all consistent with the predicted molecular weight. Then, the purified protein was analyzed by western blot. The results showed that specific bands were displayed, indicating that BL21 strain successfully expressed recombinant proteins (Figure 2B).

3.3 Establishment of indirect ELISA based on recombinant proteins

Then, we constructed an indirect ELISA method based on these recombinant proteins for the detection of porcine pseudorabies (Figure 3). The purified diluted fusion proteins (0.5, 1, 3, 5 mg/L) were coated as antigen, and the negative and positive sera were diluted at the ratio of 1:20, 1:50, 1:100, and 1:200 to optimize conditions. As shown in Table 1, the concentrations of fusion proteins gB/C and gC/D were 3 mg/L, and the sample serums were diluted at 1:50 to maximize the P/N value; the concentration of fusion protein gB/C/D was 1 mg/L, and the sample serum was diluted at 1:50 to maximize the P/N value.

3.4 Validity verification for indirect ELISA based on recombinant proteins

184 cases of PVR-positive pig sera were collected to verify the specificity of the indirect ELISA method obtained in the above experiment. As shown in Table 2, of the 184 samples, 171 were diagnosed as positive and 6 cases were misjudged as negative by IDEXX kit; 169 cases were diagnosed as positive and 6 cases were misjudged as negative by gB/C/D-ELISA; 163 cases were diagnosed as positive and 8 cases were misjudged as negative by gC/D-ELISA; 168 cases were diagnosed as positive and 7 cases were misjudged as negative by gB/C-ELISA. The positive coincidence rates of three recombinant proteins ELISA and IDEXX kit were 98.25%, 95.32%, and 98.83%, respectively; the negative coincidence rates were 85.71%, 75%, and 100%, respectively. Statistical analysis showed that there was no significant difference in the positive rate between the three fusion proteins and IDEXX kit. Importantly, gB/C/D-ELISA had the highest specificity and sensitivity among the fusion protein ELISAs developed in this study, but it still needed to be improved.

To clarify the repeatability of the ELISA established in this study, intra batch and inter batch repeatability tests were performed on five randomly selected serum samples.¹⁴ For gB/C-ELISA, the intra batch coefficient of variation (CV) was 1.8%, and the inter batch CV was 4.3%, which were less than 5%, indicating that the gB/C-ELISA method was reproducible (Table 3). As shown in Table 4, the intra batch CV was 1.8%, and the inter batch CV was 3.4%, suggesting good repeatability of gC/D-ELISA. For gB/C/D-ELISA, the intra batch CV was 1.8%, and the inter batch CV was 3.0%, which also were less than 5% and indicated a high repeatability of gB/C/D-ELISA (Table 5). Therefore, the ELISA methods established in this study had good repeatability.