J. Biomedical Science and Engineering, 2009, 2, 128-134
Published Online April 2009 in SciRes. http://www.scirp.org/journal/jbise JBiSE
Analysis and expression of the polyhedrin gene of
Antheraea pernyi nucleopolyhedrovirus (AnpeNPV)
Jia-Xi Huang1, Hui-Ling Wu1, Yan Wu1, Shan-Ying Zhu1, Wen-Bing Wang1
Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, P. R. China
Received Dec. 3rd, 2008; revised Jan. 1st, 2009; accepted Jan. 5th, 2009
ABSTRACT
The polyhedrin (polh) gene is often used to
analyse evolution of baculovirus. In this report,
the polh of Antheraea pernyi nucleopolyhedro-
virus (AnpeNPV) was cloned and sequenced.
The Open reading frame (ORF) of the AnpeNPV
consists of 738 nucleotides encoding 245 amino
acids with molecular masses of 29 kDa. The
deduced amino acids were significant homol-
ogy with other baculoviruses, such as Attacus
ricini NPV (ArNPV) and Autographa californica
NPV (AcNPV). A strongly hydrophilic region was
predicted at positions from 30 to 50 of the An-
peNPV Polh protein by bioinformatics analysis.
Expression of the polh gene of AnpeNPV in E.
coli was examined by SDS–PAGE, Western blot
and Mass-spectrum analysis. The result showed
that the bacterium expression system was
suitable for the virus gene expression. It indi-
cated that the products of the polh gene ex-
pressed in this system can be easier to use for
raising antibodies.
Keywords: Antheraea Pernyi, Insect, Baculovi-
rus, NPV, Polyhedrin, Prokaryotic Expression
1. INTRODUCTION
The Chinese oak silkworm Antheraea pernyi (Lepidop-
tera: Saturniidae) is an economically important insect
primarily for the production of tussah silk. In recent
years, consumption of the silkworm pupae as food deli-
cacies has also gained tremendous popularity. The jaun-
dice disease of the oak silkworm caused by the infection
of A.pernyi nucleopolyhedrovirus (AnpeNPV) is a major
threat to the tussah industry [1]. AnpeNPV is a member
of the Baculoviridae with large, enveloped, dou-
ble-stranded DNA. Baculoviridae are widely known to
the scientific community in the form of commercial
baculovirus expression vectors (BEVs) [2,3]. Baculovi-
ruses also have an established application as insecticides
against agricultural and forestry pests [4,5]. Currently,
the Baculoviridae comprises two genera, Nucleopolyhe-
drovirus (NPV) and Granulovirus (GV) [1]. During the
infection cycle, NPVs produce two structurally and
functionally distinct virion phenotypes: occlusion- de-
rived virus (ODV) and budded virus (BV) [6]. The oc-
cluded viruses of the NPV are referred to as polyhedra.
Polyhedrin is the major protein component of the poly-
hedra [7]. The polh gene is not essential for viral devel-
opment, and normally deletion of the polh gene is not
interfering with viral replication in cultured cells. How-
ever, in per os infectivity, the polyhedra or occlusion
bodies are required for the oral infection of insects [8].
Baculovirus entry into host cells involves that ODVs are
released from the occlusion body by the alkaline envi-
ronment within the midgut lumen of the larva and sub-
sequently initiate primary infection of the mature co-
lumnar epithelial cells of the midgut [6].
In order to explore effective propagation and infectiv-
ity of the polyhedra, this paper analysised the nucleotide
sequence and promoter (prmoter-Ap) of the polh gene of
AnpeNPV by bioinformatics tools, and further prokary-
otic expression for AnpeNPV polyhedrin (polh-Ap).
2. MATERIALS AND METHODS
2.1 Materials
The Wild-type AnpeNPV strain was maintained in our
laboratory. Restriction Enzymes, T4 DNA ligase, PCR
reagents pMD18-T and DNA purification kit were pur-
chased from TaKaRa Company (China, Dalian); primers
and other reagents were bought from Shanghai Sangon
Bio-technology Corpotation. The vectors for expression,
and Escherichia coli strain DH5α and BL21 were kept in
our laboratory.
2.2. Aplification of the AnpeNPV polh Gene
AnpeNPV genomic DNA was isolated using the method
described by previously [9,10] and about 15-20 ng DNA
was used as template for standard PCR. The specific
primers were designed based on the sequence of ORF
(GenBank: EU195295). The polh-Ap forward primer (5＇
CCG GAA TTC ATG CCA GAT TAC TCA TAC CGG
3′)containing an EcoR I restriction site (underlined), and
the reverse primer (5′ CCC AAG CTT CTA GTA CGC
GGG GCC AGT 3′) containing a Hind III restriction site
(underlined). The PCR conditions were 1 cycle at 94 °C
for 5 min; 30 cycles at 94 °C for 45 s, 62°C for 45 s, and
72 °C for 1 min; and 1 cycle at 72 °C for 10 min. The
PCR product was examined by electrophoresis in 1%
agarose gel with the ethidium bromide staining.
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129 J. X. Huang et al. / J. Biomedical Science and Engineering 2 (2009) 128-134
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2.3. Cloning and Construction of Expresion
Plasmid
The PCR products were ligated into pMD18-T vector
using T4 DNA ligase and then transformed into E. coli
(DH5a), and sequenced, respectively.
The recombinant plasmid pMD-polh-Ap was digested
with EcoR I and Hind III, and was purified to ligate with
the Pet28a vector digested with EcoR I and Hind III, and
transformed into E. coli (BL21).
2.4. Analysis of the polh Gene
The amino acid sequence was deduced with Expasy
Translate tool (http://au. expasy. org/tools/dna. html)
according to the AnpeNPV polh gene sequence. Align
using DNAstar CLUSTAL W program. Phylogenetic
tree was made by MEGA 3.1 software.
In order to explore regulatory sequence in the putative
promoter region, NNPP (Promoter Predication by Neural
Network http://www.fruitfly.org/seq_tools/promoter.html),
promoter scan and transcription factor binding sites
(http://www-bimas.cit.nih.gov/molbio/proscan/) were ap-
plied together to make a comprehensive prediction.
2.5. Expression of the polh Gene in E.coli
A positive clone was cultured in LB medium supplement
with Kanamycin (final concentration of 50μg/ ml) ove-
night at 37 ℃ with shaking, then the culture was added
into 100mL fresh LB medium and cultured at 37℃ with
shaking to A600 about 0.6. The culture was induced with
IPTG (final concentration of 8 μg/ mL) and shaked at
30 ℃ for 10 hours. SDS polyacrylamide gel was used
to analyze the expression in the Mini-Protein system
(Bio-Rad, USA). After electrophoresis, the gel was
stained with Coomassie Brilliant Blue R250 to visualize
the protein bands. Protein samples were separated on
SDS-10% polyacrylamide gels and transferred to PVDF
membranes. Blots were soaked in TBST buffer (10
mmol/L Tris-HCl, pH 7.6, 0.15 mol/L NaCl, 0.1%
Tween 20) with 5% nonfat dried milk. The antiserum
against the His-Polh fusion protein (His antibody) at a
dilution of 1:2,000 monoclonal antibody was added as
the first antibody, followed by addition of 1:5,000 dilu-
tion horseradish peroxidase-conjugated goat anti-mouse
immunoglobulin G as the secondary antibody. Blots
were visualized with the Enhanced chemiluminescence
Western blot kit (Amersham). The predicted Polh protein
band was cut out for Mass-spectrum analysis.
3. RESULTS
3.1. Nucleotide and Amino Acid Sequence
Analysis
The ORF of cloned gene has two different nucleotides
from the published sequence (DQ486030), but no amino
acid residues were changed. The 738 nucleotides (in-
cluding the stop codon TAG) encoded a putative peptide
of 235 amino acids by an Expasy Translate tool.
Figure 1. Nucleotide sequence and deduced amino acid sequence of the polyhedrin gene. The predicted amino acid is
represented by the one letter code designation below the nucleotide sequence. The initiate and the stop codes are framed.
ATGCCAGATTACTCATACCGGCCGACCATTGGTCGCACCTATGTGTACGACAACAAGTAT
M P D Y S Y R P T I G R T Y V Y D N K Y
TACAAAAACTTAGGGTCCGTCATTAAAAACGCCAAGCGCAAGAAGCATTTAGTCGAACAT
Y K N L G S V I K N A K R K K H L V E H
GAAGAGGAAGAAAAGCATTGGGATCCTTTAGACAATTACATGGTCGCGGAAGACCCTTTC
E E E E K H W D P L D N Y M V A E D P F
CTGGGGCCGGGTAAAAACCAAAAACTGACACTTTTCAAGGAAATCCGCAACGTTAAACCC
L G P G K N Q K L T L F K E I R N V K P
GACACAATGAAACTTATTGTCAACTGGAGCGGTAAAGAATTTCTGCGCGAAACTTGGACC
D T M K L I V N W S G K E F L R E T W T
CGTTTTGTTGAGGATAGCTTTCCGATTGTAAACGACCAAGAGGTCATGGATGTGTTCCTC
R F V E D S F P I V N D Q E V M D V F L
GTCATTAACCTGCGCCCCACGCGCCCCAACAGGTGCTACAAGTTCCTGGCGCAGCACGCG
V I N L R P T R P N R C Y K F L A Q H A
CTCAGATGGGACTGCGACTACGTGCCGCACGAGGTAATCCGCATTGTGGAGCCATCCTAC
L R W D C D Y V P H E V I R I V E P S Y
GTGGGCATGAACAACGAGTACAGAATTAGCCTCGCCAAGAAAGGCGGCGGCTGCCCCATC
V G M N N E Y R I S L A K K G G G C P I
ATGAACATTCACAGCGAGTACACCAACTCGTTTGAATCGTTTGTAAACCGCGTAATCTGG
M N I H S E Y T N S F E S F V N R V I W
GAGAACTTTTACAAGCCCATTGTGTACATTGGCACGGACTCGGGTGAGGAGGAGGAAATT
E N F Y K P I V Y I G T D S G E E E E I
CTCATCGAGGTTTCGCTTGTGTTCAAGGTCAAGGAGTTTGCGCCCGACGCGCCACTGTTT
L I E V S L V F K V K E F A P D A P L F
ACTGGCCCCGCGTACTAG
T G P A Y

X. Huang et al. / J. Biomedical Science and Engineering 3 (2009) 128-134 130
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The nucleotide sequence of Polh-Ap and its deduced
amino acid sequence are shown in Figure 1. This de-
duced polypeptide contains 16 strongly basic, 16
strongly acidic, 113 hydrophobic and 58 hydrophilic
amino acids with the calculated molecular mass of 29
kDa, and the isoelectric point was of 6.1.
3.2. Protein and Homology Analysis
Using BLAST software of NCBI to search for homology
in the GenBank database, the deduced amino acid se-
quence showed an identity of 97%, 98%, 98%, 97%,
93% and 89% to the corresponding genes of Attacus
ricini NPV(ArNPV, AAP16625)，Epiphyas postvittana
NPV (EppoNPV, NP_203170), Maruca vitrata MNPV
(YP_950731), Rachiplusia ou MNPV (RoMNPV,
NP_702998) [11], Bombyx mori NPV (BmNPV, AAA
46734) [12] and Autographa californica NPV (AcNPV,
NP_054037) [13], respectively. Comparison of the de-
duced amino acid sequence with that of the correspond-
ing genes of many species is shown in Figure 2. This
protein was demonstrated to be highly conserved in
baculoviruses.
The predict of secondary structure for polh-Ap by CLC
Protein Workbench 3.0.3.(Figure 4). There are 4 regions

131 J. X. Huang et al. / J. Biomedical Science and Engineering 2 (2009) 128-134
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Figure 2. Alignment of the polyhedrin genes of baculoviruses, The sequences were aligned using DNAstar CLUSTAL W program.

X. Huang et al. / J. Biomedical Science and Engineering 3 (2009) 128-134 132
SciRes Copyright © 2009 JBiSE
MacoNPV-A
PfMNPV
MacoNPV-B
EcobNPV
BusNPV
ChchNPV
TnsNPV
HearNPV
AgseNPV
SlMNPV
SfMNPV
SeMNPV
AcMNPV
HycuNPV
BmNPV
RoMNPV
AnpeMNPV
ArMNPV
AngeMNPV
EppoNPV
OpMNPV
CfMNPV
Choristoneura rosaceana
LdMNPV
0.02
Figure 3. Phylogeny of the polyhedrin protein. Phylogenetic tree of polyhedrin gene was constructed
by MEGA version 3.1 from CLUSTAL W alignments. The neighbor-joining method was used to con-
struct the tree. From the phylogenetic tree, the polh gene of AnpeNPV was closest to that of ArNPV.
Figure 4. The secondary structure of the Polh protein of AnpeNPV. It contains 4 regions of alpha helix and 11
pieces of ß-sheet.
of helical and 11 pieces of ß-sheet in the sequence.
3.3. Construction of Expression Plasmid
The fragment of polh-Ap was sequenced to be sure con-
taining a correct ORF, and was inserted into the expres-
sion pET28a vector and then was expressed in E col
(BL21) The recombinant plasmid was identified by di-
gestion with Eco R I and Hind III. The result of electro-
phoresis indicated the recombinant plasmid was suc-
cessfully constructed (Figure 6).
3.4. Expression of the AnpeNPV polh Gene
in E. coli
The E. coli BL21 transformed with the pET28a/polh-Ap
plasmid to express the His-6PGL fusion protein of about
34 kDa, which was consistent with the expected mo
lecular mass of the fusion protein of pET28a/polh-Ap
(Figure 7). The result showed that the AnpeNPV polh
gene was highly expressed in E. coli. The expression
products can be used as antigen to raise the antibody of
the Polh protein.
0.02

133 J. X. Huang et al. / J. Biomedical Science and Engineering 2 (2009) 128-134
SciRes Copyright © 2009 JBiSE
Figure 5. The hydrophobicity profile of AnpeNPV Polh protein.
The X-axis contains 245 increments, each representing an
amino acid in the sequence of AnpeNPV polyhedrin. The Y-axis
represents the range of hydrophilicity values (from 2.2 to -3.1)
with employ of Kyte-Doolittle scale. One region of strongly hy-
drophilicity exists at positions from 30 to 50 of the AnpeNPV
polyhedrin protein.
Figure 6. Identification of the recombinant plasmid pET28a/polh-
Ap by electrophoresis in agarose gel 1, pET28a/polh-Ap; M, DNA
molecular mass marker.
Figure 7. The expression products of AnpeNPV polh gene were
analyzed by SDS-PAGE and Western-blot 1, Protein of E. coli
BL21 contained pET28a induced by IPTG; 2, Protein of E. coli
BL21 contained pET28a /polh-Ap induced by IPTG; 3, West-
ern-blot of the fusion protein; M, Protein marker.
4. DISCUSSION
In this report the AnpeNPV poly gene was cloned and
compared with other baculoviruses. Polyhedrin genes
Figure 8. Mass-spectrum with Mascot analysis (Mass: 29003;
Score: 107; Expect: 0.00013) Protein score is -10*Log (P), where
P is the probability that the observed match is a random event.
Protein scores greater than 81 are significant (p<0.05).
are highly conserved among many baculoviruses. The
AnpeNPV polyhedrin gene was closest to that of ArNPV
from the Phylogeny tree (Figure 3), differing by only
five amino acids (Figure 2). DNA sequence comparison
polyhedra containing low numbers of virions [16]. of
AnpeNPV and ArNPV polyhedrins showed that a dif-
ference in identity to 97%, of which only thirteen dif
ferences (Figure 2). The result suggests that the poly
genes of AnpeNPV and ArNPV have evolved from a
common ancestor distinct from the other NPVs. The
AnpeNPV polh gene is very closely related to NPV
group I than that of group II (Figure 3). Availability of
polyhedrin protein sequences of other baculoviruses may
aid in their classification and may help define baculovi-
rus species [14].
Alignment results showed that the variability regions
occur at the beginning of N-terminus (position 2 to 4)
and the domain from position 31 to 52 (Figure 2). Even
in this region, some positions are conserved, such as,
H37, H41, E45 and D49. In contrast, the C-terminus
(from 198 to 245) is highly conserved. The cysteine
positons (at 133 and 179) and the prolines (at 60，64，81，
109，127，130，15 0，159，180，207，236，and 244)
of AnpeNPV polyhedrin appear to be very important
(Figure 2). Cysteines often form disulphide bonds criti-
cal for protein structure; proline breaks helical and
ß-sheet regions and is often associated with turns in the
secondary structure of proteins [15]. Therefore, both
these amino acids could be crucial in determining the
conformation of these proteins. These conserved regions
may be necessary to give the proteins their characteristic
common properties: namely crystal formation and alkali
solubility. Indeed, a mutant of AcNPV with a single pro
tein changed to Leu at position 62 resulted in cubic
polyhedra containing low numbers of virions [16].
Hydrophilic regions are exposed on surface of the pro-
tein and are highly polar. They have a tendency to be
antigenic sites [17]. There is one region of strongly hy-
drophilicity at positions from 30 to 50 in the AnpeNPV
polyhedrin (Figure 5). Comparison of the baculovirus
polyhedrin sequences indicates that although they vary
in amino acid sequence in this region, their basic pattern
of hydrophilicity is preserved (date no shown). There-
fore, much of the variation in amino acid sequence is
N
umber of Hits
10
5
0
50 75 100
Probabilit
y
Based Mowse Score

X. Huang et al. / J. Biomedical Science and Engineering 3 (2009) 128-134 134
SciRes Copyright © 2009 JBiSE
neutral and does not alter the overall nature of the pro-
teins. This region therefore presents a potential antigenic
site which may be useful for production of antibodies
capable of differentiating or identifying different bacu-
loviruses [18]. Ultimately predicted antigenic determi-
nants from proteins of pathogenic organisms might also
be useful in the production of synthetic vaccines [17].
The polh gene of baculovirus is a very late gene which
expressed in late stage of virus infection. It is not an
essential gene in virion development and could be de-
leted for foreign gene expression [19,20]. Some evi-
dences showed that the level of the foreign gene expres-
sion was related to genetic codes of the gene. To test the
polh gene expression in another system, we constructed
a bacterium expression system to express the Polh pro-
tein. The result indicates that this gene is suitable for E.
coli expression system. It might be helpful to produce
the virus proteins to raise antibodies.
ACKNOWLEDGEMENTS
This work was supported by the 973 National Basic Research Program
of China (2005CB121005); The Six-Field Top programs of Jiangsu
Provice; National Natural Science Foundation of Jiangsu Education
Communitte(06KJD180043); Innovation Foundation for Graduate
Students of Jiangsu Province.
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