Expression of CVN and CL7-CVN
After induction at 37 °C for 4 h, CL7-CVN was highly expressed in E. coli, and most of CL7-CVN were found in the lysate supernatant (Fig. 1b). In contrast, CVN without CL7 were basically in the lysate precipitation. The N-terminal CL7 performed as effective solubility-enhancing fusion tag and could be used for the soluble expression of CVN. To further improve the soluble expression of CVN, we preliminarily optimized the induction conditions of CL7-CVN expression (Temperature, IPTG concentration, Time). Briefly, the expression level was the higher (~ 40%) when IPTG of 0.25 mM was used to induce culture at 18 °C for 20 h (Fig. 1c).
Purification of CVN and CL7-CVN
The thermal stability of CL7-CVN was determined before protein purification. The most of proteins were denatured under heating at 100 °C, while the CVN fusion protein was intact, indicating that CL7-CVN had excellent thermal stability and could even exist at 100 °C for more than 2 h (Fig. 1d). Thus, we simplified the steps of CVN purification as described previously (Fig. 2a). In particular, the purity of CL7-CVN reached ~ 80% after the supernatant of cell lysis is heated. Besides, the purity of CL7 -CVN reached ~ 99% via additional one-step affinity purification with Ni–NTA (Fig. 2b). Moreover, 10 mg of CVN with a purity of up to 99% were obtained from 1 g of E. coli by one-step affinity purification with the digestion of HRV 3C protease (Fig. 2c). These demonstrated that CVN or CL7-CVN could be easily and economically purified using this system.
Stability analysis of CVN fusion protein
To further evaluate the stability of CL7-CVN, equal moles of CL7-CVN and CVN were respectively incubated with fresh chicken serum for 30 h at 37 °C. Western blot results demonstrated that the protein level decreased with the increase of incubation time (Fig. 2d). Moreover, CVN decreased by half when it was incubated for 24 h, yet CL7-CVN decreased by ~ 28% when it was incubated for 30 h (Fig. 2e). CL7 significantly enhances the stability of CL7-CVN in serum, which is of great significance for its application.
Analysis of antiviral activity
After efficient production of proteins, the PRV(ΔUL21/EGFP) was considered as a virus model to determine antiviral activity of CVN. PK15 cells were infected with PRV treated with CL7-CVN, CVN, and CL7 at 0.25 μM. Comparing fluorescence intensity of PK15 cells, we recognized a significant decrease in multiplication of PRV treated with CVN or CL7-CVN after 16 h of culture (Fig. 3a). Besides, we measured the mRNA of PRV gD in PK15 cells via qPCR assay. The relative virus mRNA levels of CVN and CL7-CVN groups decreased by 79.59% and 77.64%, respectively, while CL7 did not significantly affect the mRNA level (Fig. 3b).
Besides, the TCID50 assay was used to assess the effect of CVN and CL7-CVN on PRV virulence. Compared to the negative control, PRV treated with CVN at 0.25, 0.5, 1 and 2 μM resulted in 0.5, 1.63, 1.75 and 1.25 lgTCID50 reduction, respectively. Meanwhile, CL7-CVN also resulted in 0.375, 1.125, 1.375 and 1.25 lgTCID50 reduction, respectively (Fig. 3c). The results showed that purified CL7-CVN and CVN inhibited PRV from infecting PK15 cells, indicating that the prepared CVN and CL7-CVN had eminent biological activity.
Furthermore, the ELISA was also implemented to characterize the affinity of CVN to PRV. CL7-CVN could bind to PRV as well as CVN, while CL7 could not recognize and combine PRV. Besides, with the decrease of CL7-CVN or CVN concentration, the OD450 value decreased, suggesting its binding activity was dose-dependent (Fig. 3d).
Enrichment and detection of PRV
To improve the detection limit of existing PRV diagnostic assays, we explored a strategy for PRV enrichment utilizing Im7 beads with CL7-CVN. The process of virus isolation and enrichment was carried out as illustrated in (Fig. 4a). Im7 Beads treated with PRV sample and CL7-CVN had apparent fluorescence signal after washing. Conversely, the fluorescence signals of the control groups PBS, CL7, and CVN, weren’t detected (Fig. 4b). Moreover, standard PCR assays for the enriched PRV were carried out. 206 bp fragments of the gD gene, 331 bp fragments of the gB gene, were respectively amplified from the PRV captured by Im7 Beads with CL7-CVN, whereas no fragments were observed when PRV samples were treated with Im7 Beads and PBS, CL7, CVN (Fig. 4c, d).
To further validate the reliability of our enrichment method, the amount of captured PRV was determined by qPCR. The PRV enrichment efficiency was calculated as the captured viral load divided by the initial viral load before the enrichment. A series of 100 μL PRV samples (106 viruses/μL) was prepared for virus enrichment utilizing CL7, CVN, and CL7-CVN at 0.25 μM, Im7 Beads at 0.5 mg/mL. The enrichment results showed that ~ 90.78% of PRV were captured by Im7 Beads with CL7-CVN. Simultaneously, 20.93% of PRV were enriched by nonspecific physical adsorption by Im7 Beads with PBS (Fig. 5a). The efficiency of virus enrichment was calculated using different concentrations of CL7-CVN with Im7 Beads at 0.5 mg/mL. The results showed that the enrichment efficiency increased with the increase of CVN concentration in a certain range, and 97.32% of virus was captured at 0.5 μM of CL7-CVN (Fig. 5b).
Capability of PRV enrichment
To further assess the capability of our enrichment strategy, we tested the effect of sample size on the efficiency of virus enrichment. The 100 μL PRV samples (106 viruses/μL) were respectively diluted to different final volumes for PRV enrichment. When the viral sample was diluted to 1000 μL, the enrichment efficiency of virus had little change and reached 88.72% (Fig. 5c), and 467 bp fragments of the gK gene were also respectively amplified from the captured PRV (Fig. 5d). To investigate the viral load of Im7 Beads, 0.05 mg Im7 Beads immobilized with CL7-CVN was applied to concentrate PRV from the 100–500 μl samples (106 viruses/μL). When the virus sample volume increased from 100 to 300 μL (106 viruses/μL), the amount of the virus enriched increased to 3 times (Fig. 5e) and the enrichment efficiency of the virus also reached 89.64% (Fig. 5f).