J Vet Sci.  2013 Dec;14(4):441-447. 10.4142/jvs.2013.14.4.441.

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells

• Gao J
• Meng C
• Chen Z
• Li C
• Liu G

• KMID: 1712311
• DOI: http://doi.org/10.4142/jvs.2013.14.4.441

Abstract

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.

Keyword

capsid protein; codon optimization; rabbit hemorrhagic disease; Spodoptera frugiperda 9 cells

MeSH Terms

Animals
Antigens, Viral/genetics/metabolism
Caliciviridae Infections/prevention & control/*veterinary/virology
Capsid Proteins/*genetics/metabolism
Cell Culture Techniques/*methods
Codon/genetics/metabolism
Enzyme-Linked Immunosorbent Assay/veterinary
*Gene Expression Regulation, Viral
Hemorrhagic Disease Virus, Rabbit/*genetics/immunology
*Rabbits
Recombinant Proteins/genetics/metabolism
Sf9 Cells
Spodoptera
Viral Structural Proteins/*genetics/metabolism
Viral Vaccines/genetics/immunology
Antigens, Viral
Capsid Proteins
Codon
Recombinant Proteins
Viral Structural Proteins
Viral Vaccines

Figure

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  Fig. 1 Sequences of the original and codon-optimized capsid genes. Rare codons of the Spodoptera frugiperda 9 (Sf9) cells are marked with boxes.

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  Fig. 2 Detection of RHDV capsid proteins expressed in Sf9 cells by IFA. The expression of capsid (A) and opti-capsid (B) proteins was detected in recombinant rBv-Cap- and rBv-opti-Cap-infected as well as uninfected Sf9 cells (C) with anti-VP60 antiserum as the primary antibody. Scale bars = 100 µm (A~C).

• 

  Fig. 3 Western blot analysis of extracts from Sf9 cell expressing the capsid protein using a polyclonal anti-RHDV rabbit serum. Lanes A: baculovirus rBv-Cap-infected Sf9 cells, Lane B: baculovirus rBv-opti-Cap-infected Sf9 cells, Lane C: uninfected Sf9 cells, Lane M: molecular size markers (90, 66, 45, 35, 27, and 15 kDa).

• 

  Fig. 4 Electron microscopy evaluation of RHDV-VLP. The sucrose gradient fraction was applied to carbon-coated grids, negatively stained with 2% uranyl acetate, and observed at a magnification of 4,000× with electron microscopy.

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