2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

Abstract

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.

Figure 1. Cleavage mechanism and sequence analysis of 2A self-cleaving peptide.

(A) Self-cleavage mechanism of 2A self-cleaving peptide. The cleavage site locates between glycine (G) and praline (P) at its C-terminus. After self-cleavage, amino acids residues at the N-terminus of 2A linked to upstream protein and its last praline residue remained to the downstream protein, but the residues of 2A could be removed through furin and signal peptide. (B) Sequence analysis of six 2As which was respectively from porcine teschovirus-1 2A (P2A), thoseaasigna virus 2A (T2A), equine rhinitis a virus 2A (E2A), foot and mouth disease virus 2A (F2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A (BmIFV2A).

Figure 2. GSG spacer significantly improved the cleavage efficiency of 2A self-cleaving peptide in Sf9 cell.

(A) Schematic diagram of DsRed-2A-EGFP fusion gene expression vectors. BmCPV2A(30), BmCPV2A(GSG+30) and Control respectively was the native BmCPV2A, GSG spacer added BmCPV2A(30) and negative control. The skeleton of expression vector contained hr3CQ enhancer, BmAct4 promoter and Ser1PA was used by Wang (the same below). (B) The results of fluorescent signal post-transfection in the Sf9 cell. The White, Red and EGFP respectively indicated the results in the white, red and green lights (the same below). (C) Protein analysis of samples from the Sf9 cells. (D) Cleavage efficiency analysis between BmCPV2A (30) and BmCPV2A(GSG+30) in the Sf9 cell. Scale bar, 400um.

Figure 3. P2A-GSG possesses the highest cleavage efficiency to cleave DeRed-2A-EGFP fusion gene in the Sf9 cell.

(A) Schematic diagram of DsRed-2A-EGFP fusion gene expression vectors. GSG spacer was added at the N-terminus of each P2A, T2A, E2A, F2A, BmCPV2A and BmIFV2A. (B) The results of fluorescent signal post-transfection in the Sf9 cells. (C) Protein analysis of samples from the Sf9 cells. (D) Cleavage efficiency analysis of six type 2A self-cleaving peptides in the Sf9 cells. Scale bar, 400 um.

Figure 4. P2A-GSG cleaved the VgR-F-2A-EGFP fusion gene in the Sf9 cell.

(A) Schematic diagram of VgR-F, EGFP, VgR-F-P2A-EGFP and VgR-F-EGFP fusion gene expression vectors. (B) The results of fluorescent signal post-transfection in the Sf9 cells. (C) Protein analysis of samples from the Sf9 cells. (D) Protein analysis of samples from the culture mediums of Sf9 cells. Scale bar, 400 um.

Figure 5. Generation of transgenic spDsRed-P2A-spEGFP silkworm.

(A) Schematic diagram of pR-P2A-E transgenic vector. spDsRed-P2A-spEGFP was fusion gene consisted of signal peptide of Ser1 gene (sp), red fluorescent protein gene (DsRed), enhanced green fluorescent protein gene (EGFP) and P2A-GSG self-cleaving peptide. The skeleton of expression vector contained hr3CQ enhancer, Ser1-P promoter, Ser1PA, 3xp3EGFPSV40, pBacL and pBacR were used by Wang. (B) Screen for positive transgenic silkworm. (C) Microinjection analysis of the transgenic spDsRed-P2A-spEGFP silkworm. (D) Expression of fusion gene on mRNA level by real-time PCR. Ser1 is the endogenous gene, as a control and orange arrows were primers used in (A). Scale bar, 2 mM.

Figure 6. Expression of the spDsRed-P2A-spEGFP fusion gene in the transgenic silkworm.

(A,B) Fluorescent images for the middle silk grand and cocoons of the transgenic spDsRed-P2A-spEGFP silkworm. WT and pR-P2A-E were the wild type and positive transgenic silkworm. (C) Protein analysis of samples from the middle silk grand with anti-RFP, anti-GFP antibodies. (D,E) Protein analysis for the samples from the cocoons by the coomassie brilliant blue (CCB) staining and western blotting with anti-RFP, anti-GFP antibodies. Scale bar, 2 mM.