Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae

Journal of Asia-Pacific Entomology 14 (2011) 107–117

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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e

Characterization and expression of the pseudorabies virus (NYJ strain) glycoproteins in Bombyx mori cells and larvae Hyun Na Koo a, Sung Min Bae a, Jae Bang Choi a, Tae Young Shin a, Bit Na Rae Yun a, Jae Young Choi b,d, Kwang Sik Lee c, Jong Yul Roh b, Yeon Ho Je b, Byung Rae Jin c, Sung Sik Yoo e, Jae Su Kim f, Young In Kim f, In Joong Yoon e, Soo Dong Woo a,⁎ a

Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea c College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea d Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea e Choong-Ang Vaccine Laboratory, Daejeon 305-348, Republic of Korea f AgroLife Research Institute (ARI), Dongbu HiTek Co. Ltd., Daejeon 305-708, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 1 July 2010 Revised 16 August 2010 Accepted 17 August 2010 Available online 25 August 2010 Keywords: PRV NYJ strain Bombyx mori BmNPV Bm5 cells

a b s t r a c t To characterize the NYJ strain of pseudorabies virus (PRV; Alphaherpesvirus of swine) isolated from the serum of an infected swine in Korea, the nucleotide sequence of three major glycoproteins (gB, gC, and gD) was analyzed. The expression of most potent immunogenic glycoprotein (gD) was also investigated using a Bombyx mori nucleopolyhedrovirus (BmNPV) expression system. The length of the glycoprotein genes corresponding to gB, gC, and gD of the NYJ strain were 2751 bp, 1443 bp, and 1203, respectively, and their identity ranged from 94.2% to 99.8% when compared with other strains. Phylogenetic analyses using these sequences showed that the NYJ strain forms a distinct branch with high bootstrap support. A novel transfer vector (pBmKSK4) was engineered with the polyhedrin promoter of BmNPV and a 6xHis tag to express glycoprotein gD in Bm5 cells and silkworm, B. mori, larvae. The immunogenicity of recombinant gD was demonstrated by its specific detection in both Bm5 cells and silkworm larvae by porcine anti-PRV antibody. The results of this study have implications both for the taxonomy of Korean PRV strains and vaccine development. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010. Published by Elsevier B.V. All rights reserved.

Introduction Pseudorabies virus (PRV; Alphaherpesvirus of swine) causes the epidemic Aujeszky's disease (AD), an economically important disease affecting the swine industry worldwide (Bascuñana et al., 1997). The infection can occur with or without severe clinical symptoms in swine, the natural reservoir for the virus, and the virus establishes a lifelong latent infection in neuronal ganglia (Kluge et al., 1999; Mettenleiter, 2000; Pomeranz et al., 2005). Attempts to control AD in swine include vaccination with killed or live virus vaccines and passive immunization with hyperimmune serum (Hsu and Lee, 1984, Mulder et al., 1997). However, the live or killed vaccines that are most widely used have the possibility of reversion to virulence and/or latency which can decrease their protection

⁎ Corresponding author. Fax: +82 43 271 4414. E-mail address: [email protected] (S.D. Woo).

(Kluge et al., 1999; Mettenleiter, 2000; Mulder et al., 1997). Therefore, the development of DNA and subunit vaccines from immunogenic proteins which may provide safe and effective alternatives is needed (van Rooij et al., 2000; Hong et al., 2002; Shams, 2005; Yoon et al., 2006). PRV belongs to the genus Varicellovirus in the family Herpesviridae (Murphy et al., 1995). The mature virion, or infectious viral particle, consists of four morphologically distinct structural components. The central core contains the linear, double-stranded DNA genome of the virus. The DNA is enclosed in a protective icosahedral capsid to form a nucleocapsid which is embedded in a protein matrix known as the tegument. The tegument is surrounded by the envelope, a lipid membrane containing several viral glycoproteins (Mettenleiter, 2000). Among these glycoproteins, gB, gC, and gD are involved in the essential steps of PRV infection. They induce protective immune responses in hosts as noted in vaccination experiments in mouse and swine models (Hong et al., 2002; Yoon et al., 2006). Several B-cell epitopes detected on PRV gB and gC glycoproteins and T-cell epitopes detected on the PRV gC glycoprotein induce both humoral

1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2010. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2010.08.002

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and cytotoxic responses in hosts (Zaripov et al., 1998; Ober et al., 1998, 2000; van Rooij et al., 2000). Additionally, vaccination of mice and swine with either purified recombinant gD or recombinant gD-expressing virus vectors conferred protection to the animals (Eloit and Adam, 1995; Gonin et al., 1996; Monteil et al., 2000). Poxvirus, adenovirus, and herpesvirus are favored as vaccine vectors due to their ability to elicit humoral as well as cell-mediated immune responses against the inserted antigen (Shams, 2005; Dudek and Knipe, 2006; Brave et al., 2007). However, the potential disadvantages include cytotoxicity, vaccine virus multiplication in the immunized host, and pre-existing or induced immunity against the vector virus which can diminish or prevent immunity against foreign antigens (Thomas et al., 2003; Smith et al., 2005). Recombinant baculoviruses are alternative vector vaccines. The baculovirus expression system is widely used for the production of recombinant proteins, the development of subunit vaccines in insect cells (Beljelarskaya, 2002), and the generation of viruslike particles for use as vaccines (Gromadzka et al., 2006; Matassov et al., 2007). Most baculovirus expression vectors are based on the Autographa californica nucleopolyhedrovirus (AcNPV) or the Bombyx mori nucleopolyhedrovirus (BmNPV). For the mass production of foreign proteins, the BmNPV vector has a unique advantage of having an advanced system the silkworm B. mori that can be used for in vivo expression, and this system has several attractive features compared to the AcNPV vector system (Maeda, 1989; Reis et al., 1992). Although there are several PRV strains, including NYJ (Namyangju), YS (Yangsan), and IS (Iksan), in Korea, genome analysis was performed mainly on the YS strain. In this study, we describe the characteristics of Korean isolates on the molecular level through the sequence analysis of three major glycoprotein genes (gB, gC, and gD) of the PRV NYJ strain. Also, the potency of recombinant BmNPV for the development of PRV vaccine was investigated by the expression of the most potent immunogenic glycoprotein gD in both B. mori cells and larvae using novel BmNPV transfer vector system.

Table 2 PRV isolates used in this study. Virus strain

GenBank accession no.

Becker PHYLAXIA Ea NIA3 Indiana S Yamagata S-81 Kaplan Fa P-Prv FZ Min-A LA SA215 Yangsan NYJ

M17321, M12778 A25912 AF207079, AF158090 D49437 D49436 D49435 AJ271966 AY196984 EU915280 EF645837 AY169694 AY174090 DQ367438 AY217094 GQ325658, GQ325659, GQ325660, This study

Animals The larvae of the silkworm, B. mori, were F1 hybrid Baekok-Jam supplied by the Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Korea. Silkworms were reared on an artificial diet (Korea sericultural association, Korea) at 25 °C, 65 ± 5% relative humidity, and a 16-h light:8-h dark photoperiod.

Materials and methods Viruses and cells The wild type PRV NYJ strain (KCTC 11132BP), which was generously supplied by the Choong-Ang Vaccine Laboratory (Daejeon, Korea), was propagated in a porcine kidney cell line, PK-15, using Dulbecco's modified Eagle's medium supplemented with 2.5% fetal bovine serum (Gibco BRL, Paisley, UK), penicillin (100 U/ml), and streptomycin (100 U/ml). The cultures were incubated at 37 °C in a humidified CO2 incubator. The virus stocks were concentrated, titrated, and stored in aliquots at −80 °C until needed. Bm5 cells were cultured at 27 °C in TC-100 insect medium (WelGENE, Daegu, Korea) supplemented with 5% fetal bovine serum.

Table 1 Primers used for amplification of the PRV genome. Primer

Nucleotide sequencea

gB-F gB-R gC-F gC-R gD-F gD-R

5'-ATG CCC GCT GGT GGC GGT CTT-3' 5'-CTA CAG GGC GTC GGG GTC CTC-3' 5'-ATG GCC TCG CTC GCG CGT GC-3' 5'-TCA CGG CCC CGC CCG GCG G-3' 5'-T CT A GAA ATG CTG CTC GCA GCG CTA TTG G-3' 5'-CTG CAG CTA CGG ACC GGG CTG CGC TTT-3'

a

Restriction sites are underlined. The initiation codons for the each gene are shown in bold.

Fig. 1. Flow chart for the construction of the transfer vector pBmKSK4. The DNA fragment containing 6xHis-Tag, S-Tag and EK region was ligated to the transfer vector pBmKSK3.

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A

Fig. 2. Nucleotide sequence comparison of the glycoprotein genes gB (A), gC (B), and gD (C) of the NYJ strain sequenced in this study with other known PRV isolates. The start and stop codons of each ORF are shown in bold type.

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Fig. 2 (continued).

PCR

Multiple alignments and phylogenetic analyses

Viral DNA was extracted from infected cell cultures with the Viral Gene-spinTM kit (iNtRON Biotechnology, Daejeon, Korea), as recommended by the manufacturer. Primers were based on the consensus sequence of the previously sequenced PRV DNA genomes available from GenBank. PCR amplification was performed using the AccuPower PCR Premix (Bioneer Co., Daejeon, Korea) and 5% DMSO. The PCR products were isolated in 0.7% agarose gel and purified with a gel extraction kit (CosmoGenetech, Seoul, Korea). The primers used for PCR amplification are listed in Table 1.

The GenBank accession numbers of the previously sequenced PRV strains used in the sequence alignments and phylogenetic analyses are detailed in Table 2. Multiple sequence alignments were performed using the ClustalX program (Thompson et al., 1997). The percentage sequence divergences between the aligned nucleotide sequences were calculated using ClustalX. The phylogenetic unrooted and rooted trees were reconstructed on aligned nucleotide sequences by the neighbor-joining method (Saitou and Nei, 1987). The constructed neighbor-joining trees were subjected to bootstrap analysis using 1000 replicates to assess the confidence values of the virus groupings and a distance matrix was obtained from bootstrapped datasets by the Kimura method (Kimura, 1980; Felsenstein, 1985). All trees were drawn using TreeView software (Page, 1996).

Cloning and nucleotide sequencing of PRV gB, gC, and gD The purified PCR products were cloned into the T&A cloning vector (RBC Bioscience, Taiwan), and the plasmids were named pTAPRV-gB, -gC, and -gD, respectively. The positive clones were screened by agarose gel electrophoresis of the plasmids isolated with a LaboPass Mini-prep Kit (CosmoGenetech, Seoul, Korea), followed by restriction endonuclease digestion and/or nucleic acid sequencing. Restriction endonucleases were purchased from commercial sources (TaKaRa, Shiga, Japan), and used according to the supplier's instructions. Nucleic acid sequencing of the clones and/or PCR fragments was performed by SolGent Co. (Daejeon, Korea) using an ABI 3730XL Capillary DNA Sequencer. These nucleotide sequences were submitted to the GenBank database under accession number GQ325658, GQ325659, and GQ325660. The potential transmembrane domains were predicted using the TMHMM2.0 server available at http://www.cbs.dtu.dk/services/TMHMM-2.0).

Construction of the transfer vector pBmKSK4 The construction of the transfer vector pBmKSK4 is summarized in Fig. 1. To construct a 6xHis tag and polyhedrin-based BmNPV novel transfer vector, the fragment containing an EcoRI site at the 5' end and a NotI site at the 3’ end was amplified by PCR from the plasmid pBAC3 (Novagen, Darmstadt, Germany). The amplified fragment was ligated into pBmKSK3 (Choi et al., 2000) digested with EcoRI and NotI. The pBmKSK4 vector contains a 6xHis tag, an S-tag, and an EK site upstream of the MCS.

H.N. Koo et al. / Journal of Asia-Pacific Entomology 14 (2011) 107–117

B

Fig. 2 (continued).

111

112

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Fig. 2 (continued).

Generation of recombinant virus

SDS-PAGE and Western blot analysis

The pTAPRV-gD construct was digested with XbaI and PstI and inserted into the transfer vector pBmKSK4, resulting in the recombinant transfer plasmid pBmPRV-gD. Bm5 cells were co-transfected with a mixture of the purified transfer vector, bBpGOZA DNA (Je et al., 2001), and Cellfectin (Invitrogen, CA, USA) according to the manufacturer's instructions. Briefly, 100 ng of bBpGOZA DNA and 500 ng of transfer vector DNA were mixed in a polystyrene tube. Five microliters of Cellfectin was gently mixed with the DNA solution and the mixture was incubated at room temperature for 1 h. The Cellfectin-DNA complex mixture was added dropwise to the medium covering the cells while the dish was gently swirled. After incubating at 27 °C for 4 h, the medium was removed, and the cells were washed twice with fresh medium. Five days after adding the Cellfectin-DNA complex mixture to the cells, the medium containing viruses released by the transfected cells was transferred to a sterile container and stored at 4 °C. A standard plaque assay procedure was used to obtain pure viral plaques from dilutions of the media harvested from the co-transfections (O'Reilly et al., 1992). The purified recombinant BmNPV was propagated in Bm5 cells. The titer was expressed as plaque forming units (pfu) per milliliter according to standard methods (O'Reilly et al., 1992).

The prepared protein samples from cells and larvae were mixed with sample buffer, boiled for 5 min and subjected to 12% SDS-PAGE gel. For Coomassie stains, gels were washed with deionized water and stained with BioSafe Coomassie. For Western blot analysis, the proteins were transferred to a nitrocellulose membrane (Pall Corp., NY, USA). After blotting, the membrane was blocked by incubation in 5% (w/v) non-fat dry milk in TBST buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h with gentle agitation. The blot was incubated with 6xHis tag (1:1000) or porcine anti-PRV (1:100) antibodies (Choongang Vaccine Lab., Daejeon, Korea) in TBST for 1 h and washed. Subsequently, the membrane was incubated with anti-mouse and rabbit anti-pig IgG horseradish peroxidase conjugate for 30 min at room temperature. After repeated washing, the immunoreactive bands were visualized using the ECL Western Blotting Detection System (Elpis Biotech, Daejeon, Korea).

Results Nucleotide sequence analysis of NYJ gB, gC, and gD

Preparation of samples Bm5 cells were infected with wild type BmNPV or recombinant virus in a 60-mm diameter dish (2 × 106 cells) at a multiplicity of infection (MOI) of 5. At 3 days after inoculation, the culture supernatant was harvested and washed with PBS, and then used to prepare SDS-PAGE sample. Individual silkworm larvae on the first day of the 5th instar were injected with 1 × 105 pfu of wild type BmNPV or recombinant virus. At 3 to 5 days post-injection, the hemolymph and fat body were collected by cutting a caudal leg and dissection, respectively. A few crystals of phenylthiourea were added to the tubes to prevent melanization. The fat body was homogenized in 10 volumes of lysis buffer (20 mM Tris–HCl pH 7.5, 50 mM NaCl, 5% Glycerol, 0.1% Triton X-100 containing protein inhibitor cocktail (Sigma-Aldrich, USA)) for 3 min and incubated in an ice bath for 30 min. The homogenate was centrifuged at 13,000 rpm for 10 min. The supernatant contained the total protein extract. The hemolymph was centrifuged at 10,000g for 10 min to remove hemocytes and cell debris, and the supernatant was stored at −70 °C until further use.

To characterize the NYJ strain isolated from diseased piglets in Korea, we determined the nucleotide sequence of three major glycoprotein genes, gB, gC, and gD, by PCR amplification. Glycoproteins gB, gC, and gD are the major immunogenicity proteins because the antibodies they induce can neutralize PRV in vitro or in vivo (Ben-Porat et al., 1986; Marchioli et al., 1987; Zuckermann et al., 1990; Kimman, 1992; Mettenleiter, 1996). Each gene was successfully amplified from the NYJ strain by the consensus sequence primers (data not shown), and the nucleotide sequences were determined and aligned with those of other previously sequenced PRV genomes. As shown in Fig. 2, the sizes of the gB (2A), gC (2B), and gD (2C) genes are 2751 bp, 1443 bp, and 1203 bp, respectively. These gene sequences were similar to other strains but there were some additions and deletions in the sequence. The detailed sequence identity among other strains for each gene is summarized in Table 3. The identities between NYJ and other PRV strains ranged from 94.2% to 99.8% for the nucleotide sequences and from 91.5 to 99.0% for the amino acid sequences.

H.N. Koo et al. / Journal of Asia-Pacific Entomology 14 (2011) 107–117

C

Fig. 2 (continued).

113

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Fig. 2 (continued).

To establish the genetic relationships of the sequenced NYJ strain, phylogenetic analyses were performed on the gB, gC, and gD nucleotide sequences. NYJ gB, gC, and gD sequences were most closely related to the Becker strain, Indiana S strain, and Kaplan strain, and most distantly related to Ea strain, P-Prv strain and LA strain, respectively (Fig. 3). The trees were supported by high bootstrap values. The same topography and high bootstrap numbers were obtained when the trees were constructed using the amino acid sequence (data not shown).

constructed using BmNPV polyhedrin gene and was used to generate recombinant virus (Fig. 1). The recombinant BmNPV, rBmPRV-gD, was constructed with the glycoprotein gD which is one of the most potent immunogenics of PRV infection (Marchioli et al., 1987). Expression of the recombinant gD in Bm5 cells was analyzed by SDSPAGE and Western blotting. The detection of the recombinant gD protein band was difficult on SDS-PAGE (Fig. 5A), but its expression was shown on Western blot analysis with 6xHis tag (Fig. 5B) or porcine anti-PRV antibodies (Fig. 5C). In silkworm larvae, recombinant gD protein could be also detected by both 6xHis tag and antiPRV antibodies in the fat body of silkworm larvae (Fig. 5D–F).

Prediction of transmembrane helices in glycoproteins gB, gC, and gD

Discussion

Most membrane proteins have hydrophobic regions which span the hydrophobic core of the membrane bilayer, and hydrophilic regions, which are located on the outside or the inside of the membrane. As glycoproteins gB, gC, and gD are membrane proteins, the putative transmembrane helices were predicted using TMHMM through the web server. TMHMM uses a hidden Markov model (HMM) to predict transmembrane helices from the primary protein structure. The results show that gB, gC, and gD contain one (amino acids 802–824), two (amino acids 7–29 and 453–475), and one (amino acids 352–374) transmembrane domains, respectively (Fig. 4).

Comparative molecular epidemiology of a virus is valuable for developing a national control and prevention strategy for the virus and disease. The analysis of molecular and phylogenetic properties of NYJ gB, gC, and gD genes clearly indicates that the NYJ strain is a novel isolate and is distinct from other Korean isolates (Table 3, Fig. 3). Interestingly, in the sequence of gD, the NYJ strain was more related to the Kaplan strain than to the Yangsan strain, a Korean isolate (Hyun et al., 1996). In this study, we could not compare more Korean strain sequences because they are not well studied. Our results indicate the necessity of more molecular research on Korean PRV strains to control AD effectively. This information also would be useful for choosing developed vaccines from various strains to control AD in Korea and in developing a Korean PRV vaccine. The glycoproteins of PRV mediate the adsorption and penetration of virus and represent a major target for a neutralizing antibody (Eloit et al., 1988). Of the eleven membrane genes coding for glycoproteins, gD is one of the most potent immunogenics of PRV infection (Marchioli et al., 1987) because it is a target of neutralizing antibodies that can protect mice and swine from PRV (Wathen et al., 1985). In addition, vaccination with recombinant gD has been shown to protect animals from PRV (Marchioli et al., 1987; Riviere et al., 1992). Although several attempts for the expression of glycoprotein gD by baculovirus were conducted, its system were unsuccessful and were evaluated only with AcNPV (Hink et al., 1991; Prud'homme et al., 1997; Grabowska et al., 2009). In this study, therefore, the expression of gD was investigated using BmNPV which can utilize both cell lines and B. mori larva. The result was not notable in the production of recombinant gD in Bm5 cells and it was detected only on Western blot analysis with 6xHis tag or porcine anti-PRV antibodies (Fig. 5). However, this suggests that pBmKSK4, a novel transfer vector constructed in this study, is very reliable for recombinant protein detection using 6xHis tag and that the recombinant gD has immunogenicity to porcine PRV serum. The low

Phylogenetic analysis of NYJ gB, gC, and gD sequences

Expression of the glycoprotein gD To evaluate the expression of recombinant PRV glycoprotein in B. mori cells and larvae, the novel transfer vector pBmKSK4 was

Table 3 Comparison of nucleotide and amino acid sequence identities (%) of the NYJ gB, gC, and gD genes with those of other PRV strains.

gB 2751 bp 916 aa

gC 1443 bp 480 aa

gD 1203 bp 400 aa

Becker

Ea

PHYLAXIA

98.6% 97.3%

97.8% 95.3%

98.5% 97.3%.

Becker

Ea

Indiana S

NIA-3

P-Prv

Yamagata S-81

98.5% 97.5%

94.6% 91.5%

98.8% 98.3%

98.6% 97.7%

94.2% 91.5%

98.6% 97.5%

Fa

FZ

Kaplan

Min-A

LA

SA215

Yangsan

98.4% 97.0%

98.9% 97.0%

99.8% 99.0%

98.8% 96.8%

98.3% 96.5%

98.2% 96.5%

98.2% 96.5%

H.N. Koo et al. / Journal of Asia-Pacific Entomology 14 (2011) 107–117

A

B

115

Ea

Ea

P-PrV Becker NYJ

NIA 3 Indiana S

PHYLAXLA

Yamangta NYJ

Becker

0.001

C

0.002

LA Fa SA215 Min-A Yangsan Kaplan NYJ FZ 0.002

Fig. 3. Phylogenetic trees based on the nucleotide sequence of the glycoprotein genes gB (A), gC (B), and gD (C) of the NYJ strain. The tree was constructed using the neighbor-joining method based on genetic distances calculated by Kimura's two-parameter method. The reliability of the tree was assessed by bootstrap analysis with 1000 replications. Scale bars at the bottom of each tree represent the number of nucleotide substitutions per site. The references for the sequences of PRV isolates used in the phylogenetic analyses are cited in Table 2.

A

TMHMM posterior propability for sequence 1.2

probability

1.0 0.8 0.6 0.4 0.2 0 0

B

100

200

300

50

100

150

400

500

600

700

800

900

1.2

probability

1.0 0.8 0.6 0.4 0.2 0 0

C

200

250

300

350

400

450

1.2

probability

1.0 0.8 0.6 0.4 0.2 0 0

50

100

150 200 250 a.a. position

300

350

level expression of gD by BmNPV was similar to previous reports of recombinant gD using AcNPV (Hink et al., 1991; Prud'homme et al., 1997; Grabowska et al., 2009). This may be concerned with the presence of a transmembrane domain in gD (Fig. 4). Most membrane proteins have hydrophobic regions which span the hydrophobic core of the membrane bilayer and hydrophilic regions located on the outside or the inside of the membrane. The cell-based expression of these membrane proteins is difficult (Katzen et al., 2009). In silkworm larvae, recombinant gD protein was detected mainly in the fat body which is important in protein synthesis and is the major site for virus proliferation. In most cases of expression of heterologous proteins in silkworm larvae, the foreign proteins are secreted into the hemolymph which facilitates the rapid extraction of proteins (Wu et al., 2001). In our experiment, however, only a small amount of recombinant protein was detected in the hemolymph (data not shown). These results indicated that recombinant gD may be associated with fat body cells rather than being secreted into the hemolymph and this is supported by the presence of a transmembrane domain within gD. Recombinant protein expression systems using insect larvae were adopted in this study because they can produce modified proteins similar to those produced in the original host cells in relatively large amounts. Unfortunately, gD was not expressed at high levels in silkworm larvae compared to Bm5 cells. Studies for modification of expressed domains and expression systems will be further required to enhance the production of recombinant gD in silkworm larvae. We are currently investigating the enhanced production of recombinant gD by the expression on the outside or inside surfaces of cell membranes which may be useful for the development of new, safe, effective and economical vaccines against PRV in Korea.

400 Acknowledgments

Fig. 4. TMHMM topology prediction and probability profile for NYJ glycoproteins gB (A), gC (B), and gD (C). The top line shows the predicted topology with predicted transmembrane helices. The thin black and gray curves show the posterior probabilities for the inside and outside loops, respectively. The striped profile shows the probability for a transmembrane helix.

This work was supported by a grant (Project No. 20070401034 002) from BioGreen 21 Program, Rural Development Administration, Korea.

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Fig. 5. Expression of the NYJ glycoprotein gD in Bm5 cells (upper panel) and the fat body of B. mori larvae (lower panel). Bm5 cells were infected at an MOI of 5 with each virus and harvested 3 days post-infection. After injection of silkworm larvae on the first day of the 5th Instar with each virus, the fat bodies were collected by dissection 4 days post-injection. Protein samples from cells or larvae were analyzed by SDS-PAGE (A and D) and western blot analysis with 6xHis tag (B and E) and porcine anti-PRV (C and F) antibodies. The recombinant gD proteins are indicated with arrowheads.

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