Design of fusion protein for efficient preparation of cyanovirin-n and rapid enrichment of pseudorabies virus

Abstract

Objective: Cyanovirin-N (CVN) is a cyanobacterial protein with potent neutralizing activity against enveloped virus. To achieve the economic and functional production of CVN, the CVN N-terminally fused with CL7(A mutant of the Colicin E7 Dnase) was utilized to improve the solubility and stability of CVN fusion protein (CL7-CVN). Additionally, to improve the detection limit of existing PRV diagnostic assays, CL7-CVN was used for Pseudorabies virus (PRV) enrichment from larger sample volumes.

Results: CVN fused with CL7 was efficiently expressed at a level of ~ 40% of the total soluble protein in E. coli by optimizing the induction conditions. Also, the stability of CVN fusion protein was enhanced, and 10 mg of CVN with a purity of ~ 99% were obtained from 1 g of cells by one-step affinity purification with the digestion of HRV 3C protease. Moreover, both purified CVN and CL7-CVN could effectively inhibit the infection of PRV to PK15 cells. Considering the bioactivity of CL7-CVN, we explored a strategy for PRV enrichment from larger samples.

Conclusions: CL7 effectively promoted the soluble expression of CVN fusion protein and improved its stability, which was meaningful for its purification and application. The design of CVN fusion protein provides an efficient approach for the economical and functional production of CVN and a new strategy for PRV enrichment.

Keywords: Anti-PRV; CL7; CVN; Rapid purification; Soluble expression; Virus enrichment.

Fig. 1

The design and expression of CVN fusion protein. a Structure designs of CVN fusion protein for the expression of His-CVN and CL7-CVN. b The soluble expression of CL7-CVN in E. coli. c Optimization of CL7-CVN expression condition (Temperature, IPTG, Time).wcl, whole cell lysates; sup, supernatant of cell lysates; pel, pellet of cell lysates (pel). d Changes of supernatant protein in cell lysates treated at 100 °C for 0–120 min

Fig. 2

Protein purification. a one-step affinity purification process for CVN and CL7-CVN. b, c One-step affinity purification of CL7-CVN and CVN. Sup, supernatant of cell lysates; H-Sup, supernatant of cell lysates heated; column, CL7-CVN on column; Cleavage, cleavage of CL7-CVN on the column; FT, flow through; EL, eluate; M, protein marker. d Western blot was used to detect the changes of CVN and CL7-CVN in serum. e The relative levels represent quantification of d using Image J software

Fig. 3

Activity analysis of purified CVN and CL7-CVN. a Fluorescence microscopy images of PK15 cells infected by PRV treated with CL7-CVN, CVN, CL7 after culturing for 16 h. b The relative levels of mRNA of PRV in the infected PK15 cells treated with CL7-CVN, CVN, CL7 (*p < 0.01). c TCID50 assay of antiviral activity of CVN or CL7-CVN. d ELISA was performed to analyze the affinity of CVN and CL7-CVN to PRV

Fig. 4

The enrichment and detection of PRV. a Schematic of the strategy for the enrichment and detection of enveloped viruses using CL7-CVN with Im7 Beads. b The fluorescence intensity of PRV captured by Im7 Beads with PBS, CL7, CVN, and CL7-CVN. 1, Im7 Beads; 2, PRV sample; 3, PRV sample reacted with Im7 Beads and PBS; 4, PRV captured by Im7 Beads with PBS after washing; 5, PRV sample reacted with Im7 Beads and CL7; 6, PRV captured by Im7 Beads with CL7 after washing; 7, PRV sample reacted with Im7 Beads and CVN; 8, PRV captured by Im7 Beads with CVN after washing; 9, PRV sample reacted with Im7 Beads and CL7-CVN; 10, PRV captured by Im7 Beads with CL7-CVN after washing; c, d PCR analysis of PRV gene (206 bp fragment of gD gene, 331 bp fragment of gB gene) captured by Im7 Beads with PBS, CL7, CVN, and CL7-CVN; PRV, positive control; Water, negative control

Fig. 5

Analysis of the enrichment capacity of PRV. a Different proteins were used to enrich virus samples (*p < 0.01). b Effect of CL7-CVN concentration on the enrichment efficiency. c Effect of sample size on the enrichment efficiency. d PCR analysis of 467 bp fragments of gK gene of PRV captured by Im7 Beads with CL7-CVN. e Viral load analysis of Im7 Beads. f Changes in enrichment efficiency of PRV in increasing sample volume (10⁶ viruses/μL)