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Bacuolvirus Expression Vectors,
BEVs
Baculovirous
nuclear
polyhedrosis virus, NPV
granulosis virus, GV
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jaundice
polyhedrin,
Maeda
. 1985 BmNPV
-interferon
5*10 units ~50 g
α- α7
μ
1 2 3���������� � ���� �����
*�����
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2: 01-12 2011( )Mdares Bulletin
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B hepatitis B
virus surface antigen, HBsAg
1989 Higashihashi ., 1991
Ho ., 1998
Li ., 2008
Miller,
1988 NPV
BmNPV
Maeda, 1989 Li ., 2008
100-1000 Kato .,
2009 Li ., 2008 Maeda, 1989
1988
136
red fluorescence protein,
RFP
Baird ., 2000
et al
et al
et al
et al
et al
et al
Discosoma genus
et al
rfp
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Royalisin
51
3
Royalisin
2006 Fujiwara ., 1990 Bachanová
. 2002 Royalisin
Apisimin Bíliková .,
2002 54
Apisimin
MRJP1
350 kDa 2007
Apisimin
Royalisin
Apisimin
et al
et al
P. l. larvae
et al
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7 N��¯OP <=�°。
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F
5
F
nuclear polyhedrosis virus, BmNPV
Royalisin Apisimin
OJ03×OJ04
4 5
5 1×10 pfu/ml
rfp
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6
1
6
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l
l
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4
RFP
RFP
Anthos Zenyth 3100, Bio-RAD Co.
535/595 nm /
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2001 Molecular Cloning
SDS-PAGE
Western Blot
12 20 SDS PAGE RFP
Royalisin Apisimin 60
120 Bio-Rad
Co.
PVDF Bio-Rad Co.
1 5000 RFP Royalisin
Apisimin
X
1-2
72
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(~ijl ô�)=Hÿ¬Q,�
% 。By,ijl1 ~�d{
|,U�S�åijl1 (�2)。
96
71 5
6
Fig. 1. The symptoms of silkworm infected with recombinant BmNPV harboring rfp.
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Fig. 2. Investigation of days after infection in various silkworm varieties.
�2 ���Ùijl¬Q=H üý。
100
80
60
40
20
01 2 3 4 5 6
���Ù(°)
ijl d(n)
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|,}�¬Q=H ,ÁÂe±�M²
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=K�þ"ìe��å���(��),
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80
������� ��� 5
M 1 2 3 4 5 6 7 8
kD
250
150
100
75
50
37
25
20
Fig. 3. Expression of recombinant RFP with BmNPV in silkworm hybrids. Lane M, molecular weight
marker. Lane 1-7, haemolymphs from the silkworm hybrids infected with BmNPV harboring .
Lane 8, haemolymphs from the silkworm uninfected with BmNPV. Arrow shows RFP with 29 KDa.
rfp
�� ��Ù��M² JK:p。1 ö,OPËt®R5��,1 ö,
,ij ��M²,1 ö,x,ij ��M² ���S ,�m� 。
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8 RFP( )
OJ03×OJ04 3.5-4
4-5
RFP
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60 2
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( )。�(� )*��
þ + � 、 、
!j�JKcR (�� 1
ö)。
Baird ., 2000
OC05×FJ01 OJ03×OJ04
OC05× RFP
1-3
et al
J� �»¼S=��ijÿe JK:pRFP
Table 1. Infected rate and production of RFP in silkworm hybrids
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、 、 !jJK.º,
t�� 、 e �ÂÃ
�。ɧsíEÛ;ÜÝ� OPË(
���/=。
OJ03×OJ04 OC05×
52.15 49.78 37.80
Fig. 4. ELISA analysis of DsRed protein using BmNPV in silkworm larvae.
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49.78
60.00
50.00
40.00
30.00
20.00
10.00
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37.80
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rescen
ce
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U
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% eÛ;ÜÝéÀsí:
Royalisin Apisimin
TC-100 100
OJ03×OJ04
5
4
20 SDS-PAGE
���7 e ,hQ
ôEñ��S �,m e
(�5,1 ö;�5,1 ö),
y�xij ��M²�éÀJK(�
7e5,1 ö)。Bñ��S8-
�R,�����ð�7 、
m� E 。
Royalisin Apisimin
6.7 5.5 kDa
1-4 1-4
N
Royalisin
Apisimin 0.3 4 mg
������� ��� 7
Fig. 6. The immunoblotting analysis of the Apisimin peptide by silkworm larvae. Lane N, haemolymphs
from the silkworm uninfected with BmNPV. Lane P, Apisimin peptide as a positive control (1 g/ l).
Lane 1-4, haemolymphs from the silkworms uninfected with BmNPV harboring Apisimin gene.
Lane M, molecular weight markers.
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S ;1 ö,,ij ��M²;1 ö,OPËt®R5��。
N P
1-4 M1 g/ l� ( )μ μ
10
N P M 1 2 3 4
M N P 1 2 3 4
Fig. 5. The immunoblotting analysis of the Royalisin peptide by silkworm larvae. Lane M, molecular
weight markers. Lane N, haemolymphs from the silkworm uninfected with BmNPV. Lane P,
Royalisin peptide as a positive control (1 g/ l). Lane 1-4, haemolymphs from the silkworms
uninfected with BmNPV harboring Royalisin gene.
μ μ
�7 1 ö,OPËt®R5��;
1 ö,xij ��M²;1 ö,ñ��S( )。
��� Royalisin �6789)*。 M
N P 1 g/ lμ μ
7.9
3.2
% &
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í<=-�78���7 �¯OP,
Maeda 1985
B
hepatitis B virys surface antigen,
HBsAg 1989 Higashihashi .,
1991 Ho ., 1998
Li ., 2008
RFP DsRed
cotransfection
Bm cell line
4
F
et al
et al
et al
rfp
rfp
α
1
�����<- �°,? ��7
�¯OP�7 C'。���?;�
Å �Ù»¼R§ Å
� S=,��ö;�(¾、��。å
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I1,ijl©ªeüý��ij:
p,T�°l1 Ê���M²,
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N �°�h4��。����
�sí,S= e
7¼-�,��Q,�ð�R ,i
jÿNñ %(V)Bë,� 7 R
N,'NT�q�,þ7 �°。
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¡W&X/0µ7¥Y�8Ë,�},
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hTUíZ[\( )。
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d_`ae�Ö。"I�ë���L
µ、]{P^Heh�k�H�H¶。
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( )。`�./,}
c "�B:r�,��OP��OP
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S",*]x�,a&��{e=
��o。¦�,�TQÕFÖYJK(u
F
OJ03 5 OJ03×OJ04 6
F 5
4-5
SDS-PAGE
RFP
OC05×FJ01 OC05×HJ04
2.3g
95 RFP
Porcine Lactoferrin Royalisin
Apisimin Royalisin
defensin
51 6
cysteine 3
Fujiwara .,1990
Royalisin
Apisimin
Bachanová ., 2002
Major protein 1, MRJP1
rfp
et al
et al
�、������������ �
1
1
�������� ���� ���8
���。 。E�¡�bMOPËc
ÏÙ�7 �OPµÖOPË
。#$���d : 。
2006
Royalisin 8 43-50
2002.
Identification of honeybee peptide
active against Paenibacillus larvae
larvae through bacterial growth-
inhibition assay on polyacrylamide
gel. INRA/DIB-AGIB/EDP Sci. 259-
269.
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DsRed, a red fluorescent protein from
coral. PNAS 97:11984-11989.
2002. Apisimin, a new serine-valine-
rich peptide form honeybee (
L.) royal jelly: purification
and molecular characterization. FFBS
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Purification and determination of the
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Ts i e n .
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Saenger, J. Klaudiny, and J. Šimúth.
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K o b a y a s h i .
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Horiuchi, Y. Saeki, K. Sakano, Y.
Sato, K. Takeda, S. Takashina, and
T. Takahashi.
Apis
mellifera
Å, )。�},����DB} °
OP�5� 8,B��78���7
�#ef�q*+JK e
。x }�#$?��T��
¢X8 ���Z8�7 ,¦.�B
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,Ë���h¢ �,��i��S�
!��Z��。
����sí:B �5���
,;�<=7 �¯OP�°,B¦.
���Ù( )�, !j
、 ��Q��,ijÿN
,7 OP 、
,�>�7 C'。���7 µÖ
e ,hQôt��
、 ,�%&Qô'=。
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!ùúZl9mnop,����kq
�ÿrhsôteuvs�7wp,x
}�yz{。
。 。
Y|I� ���}~e"
QÕFÖo�Jñ。����15
�I��� ���Z�j1
�。
。 。��bcdMHI,
LM UÃ。�AY�Q«�Ç�
j。
2007 2
Royalisin
Apisimin
OJ03×OJ04 OC05×
OC05×FJ01
RFP 28.50 32.20 g/larvae
Royalisin Apisimin
6.7 5.5kDa
2007
Apisimin
43-46
1989
rfp
' (
)�*+
���、�、� 、��
���
μ
������� ��� 9
expression and characterization of
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E . Y. P a r k .
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2009 12 31
2010 06 08
α
� �: � �
�J �: � �
�������� ���� ���10
Study on the Production of Foreign Proteins
in Silkworm ( L.)Bombyx mori
ABSTRACT
In order to produce high economic proteins using Bacuolvirus expression vectors (BEVs)
in silkworm larvae. We injected recombinant virus (vBmpDsRFP) expressing the red
fluorescent protein (RFP) to 8 silkworm hybrids which the OJ03×OJ04 been a positive
control. The primary results showed that OC05×FJ01, OJ03×OJ04 and OC05×YINFU that the
fluorescent value in the hemolymph were 52.2, 49.8 and 37.8 unit, respectively, were the best
potential candidates for producing RFP. The BEVs could also produce antimicrobial peptides,
Royalisin and Apisimin from honeybee using silkworm larvae, and Apisimin were 4 mg/
larvae in the hemolymph.
silkworm ( L.), nuclear polyhedrosis virus, baculovirus expression
vectors, antimicrobial peptides
Key words: Bombyx mori
*Corresponding author, e-mail: [email protected]
Chin-Hsun Liao *, Yun-Chen Chan , Den-Jen Wu , Mei-June Lu ,
Tzong-Yuan Wu , and Yu-Chan Chao
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Miaoli District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan,
Miaoli, Taiwan, R. O. C.
Departmant of Bioscience Technology, Chung Yuan Christian University, Chung Li, Taiwan, R. O. C.
Institute of Molecular Biology, Academia Sinica, Taiwan, R. O. C.
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