* 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, d$ e , 、 、 ,...

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* 1 1 1 1 2 3 ( ), 。, ,。, Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01 OJ03×OJ04 OC05× 52.2 49.8 37.8 4 mg 5 ( ) ,, ,。 ( )。 ( ) 。( ) ( )。, Bombyx mori L. Bacuolvirus Expression Vectors, BEVs Baculovirous nuclear polyhedrosis virus, NPV granulosis virus, GV ( ) ,( ), ,, ( ) ,。, ( ) ( ), ( ) Bombyx mori polh p10 et al nuclear polyhedrosis virus, BmNPV 5~6 NPV jaundice polyhedrin, Maeda . 1985 BmNPV -interferon 5*10 units ~50 g α- α 7 μ 1 2 3 * e-mail: [email protected] 1 2: 01-12 2011 ( ) 2: 01-12 2011 ( ) Mdares Bulletin

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Page 1: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

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Bombyx mori L.

Bacuolvirus Expression Vectors,

BEVs

Baculovirous

nuclear

polyhedrosis virus, NPV

granulosis virus, GV

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polh p10

et al

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polyhedrosis virus, BmNPV

5~6 NPV

jaundice

polyhedrin,

Maeda

. 1985 BmNPV

-interferon

5*10 units ~50 g

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μ

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e-mail: [email protected] ������� ��� 1

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2: 01-12 2011( )Mdares Bulletin

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9:e���°(Ù),�}34B -

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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KLM,�ÿS�° °��6

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Royalisin

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3

Royalisin

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. 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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�� !"

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F

5

F

nuclear polyhedrosis virus, BmNPV

Royalisin Apisimin

OJ03×OJ04

4 5

5 1×10 pfu/ml

rfp

Bombyx

mori

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1

6

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μ

l

l

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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

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72

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Page 4: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

�������� ���� ���4

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üýþg:É��°Ù ¬Q=H �

ijl1 ~,=K,Bijl13 4

(~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.

�� >���ijV �SHI =H:p。rfp

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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Page 5: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

������� ��� 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� 。

RFP M 1-7

8 RFP( )

OJ03×OJ04 3.5-4

4-5

RFP

12.3-32.2 g

Tijl ~QR=H

�{|,h���ijl �=

H。 7 R��Ùy!,�ð��

� R (J�)。μ

�üýet¹sí,���B

-�5� 8�,78ij��l,¦

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RFP

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60 2

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P( )\�

t¹,síþgÉ�>���ð�JK

�SOP,ht®RQô�

(���m�g ),�%&Qô'=

red fluorescence protein, RFP

RFP 29 KDa

( )。�(� )*��

þ + � 、 、

!j�JKcR (�� 1

ö)。

Baird ., 2000

OC05×FJ01 OJ03×OJ04

OC05× RFP

1-3

et al

Page 6: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

J� �»¼S=��ijÿe JK:pRFP

Table 1. Infected rate and production of RFP in silkworm hybrids

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\Rsí(� ),þg:��Ù

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ELISA

RFP

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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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Page 7: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

�、������������ �

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HI1B Uçè2�²3ã

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T �­� ,t�78}�°�SH

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% eÛ;ÜÝéÀsí:

Royalisin Apisimin

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OJ03×OJ04

5

4

20 SDS-PAGE

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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.

�5 ��� Apisimin �678?)*。1 ö,xij ��M²;1 ö,ñ�

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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I7²HIBJäK( );

91q�SHI。UÃ1q��SHI

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ijl1 t�ÊLwx²,�öÛ

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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'。���?;�

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���öZB7 ���'µÖóí�

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P ( )、 e

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d_`ae�Ö。"I�ë���L

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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

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Page 9: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

���。 。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.

2 0 0 0 . B i o c h e m i s t r y ,

mutagenesis, and oligomerization of

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

528:125-129.

1 9 9 0 . A p o t e n t

antibacterial protein in royal jelly.

Purification and determination of the

primary structure of royalisin. J. Biol.

Chem. 265:11333-11337.

1991. High-level

B a c h a n o v á K . , J . K l a u d i n y, J .

Kopernický, and J. Šimúth.

Baird, G. S., D. A. Zacharias, and R. Y.

Ts i e n .

Bíliková, K., J. Hanes, E. Nordhoff, W.

Saenger, J. Klaudiny, and J. Šimúth.

Fujiwara, S., J. Imai, M. Fujiwara, T.

Yaeshima, T. Kawashima, and K.

K o b a y a s h i .

Higashihashi, N., Y. Arai, T. Enjo, T.

Horiuchi, Y. Saeki, K. Sakano, Y.

Sato, K. Takeda, S. Takashina, and

T. Takahashi.

Apis

mellifera

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OP�5� 8,B��78���7

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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

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Page 11: * 1 11123 · 2012. 9. 3. · 1 11123 ( ), 。, - ./ 。 ,。, D$ E , 、 、 , "#NO56$ 、 P 。 , 。 Bacuolvirus Expression Vectors, BEVs OJ03×OJ04 8 4 OC05×FJ01

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

1 1 1 1

2 3

1

2

3

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.

11