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Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm

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Abstract

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease affecting young chickens and causes serious economic losses to the poultry industry worldwide. Development of subunit vaccine using its major caspid protein, VP2, is one of the promising strategies to protect against IBDV. This study aim to test the feasibility of using silkworm to produce recombinant VP2 protein (rVP2) derived from a very virulent strain of IBDV (vvIBDV). A total of 16 transgenic silkworm lines harboring a codon-optimized VP2 gene driven by the sericin1 promoter were generated and analyzed. The results showed that the rVP2 was synthesized in the middle silk gland of all lines and secreted into their cocoons. The content of rVP2 in the cocoon of each line was ranged from 0.07 to 16.10 % of the total soluble proteins. The rVP2 was purified from 30 g cocoon powders with a yield of 3.33 mg and a purity >90 %. Further analysis indicated that the rVP2 was able to tolerate high temperatures up to 80 °C, and exhibited specific immunogenic activity in mice. To our knowledge, this is the first report of overexpressing rVP2 in the middle silk gland of transgenic silkworm, which demonstrates the capability of silkworm as an efficient tool to produce recombinant immunogens for use in new vaccines against animal diseases.

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Acknowledgments

We are grateful to Professor Hongjuan Cui of Southwest University, China, for improving this manuscript. This work was supported by the National Basic Research Program of China (2012CB114600), and Grant (XDJK2014B014) from the Fundamental Research Funds for the Central Universities.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China

    Hanfu Xu, Lin Yuan, Feng Wang, Yuancheng Wang, Riyuan Wang, Chunnuan Song, Qingyou Xia & Ping Zhao

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  1. Hanfu Xu
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  2. Lin Yuan
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  3. Feng Wang
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  4. Yuancheng Wang
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  5. Riyuan Wang
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  6. Chunnuan Song
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  7. Qingyou Xia
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  8. Ping Zhao
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Corresponding author

Correspondence to Hanfu Xu.

Additional information

Hanfu Xu and Lin Yuan have contributed equally to this work.

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11248_2014_9827_MOESM1_ESM.tif

Fig. S1. Structure of the expression vector pBhSer1VP2. The resulting vector is composed of five units between the right- and left-arms of piggyBac transposon: BmNPV hr3 enhancer, Ser1 promoter, coding sequence of VP2, 3’-UTR sequence of Ser1, and 3×P3 promoter-driven EGFP cDNA with a SV40 polyA signal

11248_2014_9827_MOESM2_ESM.tif

Fig. S2. Generation of the transgenic silkworm positive for EGFP. (A) Bright-field (left) and fluorescent (right) images of Day-7 eggs (a, b) and an adult (c, d) that carry the 3×P3-EGFP and VP2 gene. Arrowheads indicate EGFP-positive eggs. (B) Southern blotting analysis of transgenic lines. Genomic DNA samples from transgenic (L2, 6, 7, 8, 13, 14) and wild-type (WT) moths were digested using the EcoR I and Xba I, separated by agarose gel electrophoresis, and subjected to Southern blotting using a digoxigenin-labeled probe specific for the EGFP(TIFF 3054 kb)

(TIFF 1303 kb)

11248_2014_9827_MOESM4_ESM.tif

Fig. S3. Determination of the peptide sequence of rVP2. Sequences highlighted in black indicate the original peptide sequence of VP2 in GenBank (accession no. JF907703). Blue letters show the peptide sequences of rVP2 determined by MS+MS/MS. Red letters show the mutant amino acids of VP2 (B87 strain, accession no. DQ202329). Red frame represents the neutralizing antibody binding region (TIFF 1718 kb)

11248_2014_9827_MOESM5_ESM.tif

Fig. S4. AGP analysis of rVP2 (a) and IBD antigen (b). 0: rVP2 protein; 1: PBS buffer (pH 7.4); 2: rVP2 serum; 3: B87 vaccine serum; 4: IBD positive serum; 5: IBD antigen(TIFF 3284 kb)

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Xu, H., Yuan, L., Wang, F. et al. Overexpression of recombinant infectious bursal disease virus (IBDV) capsid protein VP2 in the middle silk gland of transgenic silkworm. Transgenic Res 23, 809–816 (2014). https://doi.org/10.1007/s11248-014-9827-7

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