Expression of FMDV Expression of FMDV CapsidCapsid Proteins in Proteins in SilkwormSilkworm--BaculovirusBaculovirus Expression system Expression system
and Its Utilization as a Subunit Vaccineand Its Utilization as a Subunit Vaccine
Biotechnology Research Institute, Lanzhou Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing, China
Post Code: 100081, Tel: +86-10-68919854, Fax: +86-10-68975136
E-mail: [email protected]
Naming of The Naming of The BaculovirusBaculovirus The name of the Baculovirus comes from Greek.
“baculo” means stick in Greek. The Baculoviridae includes two genus:
Nucleopolyhedrovirus The another genus: Granulovirus. The genomic DNA of the Baculovirus was
supercoiled double strands circular DNA, about 80~180Kb in size. So, It can be expressed large foreign DNA fragment(~10kb) in this system.
1. Accommodate the large foreign gene fragment (~10Kb) .
2. ploh and p10 are both unnecessary genes of the Baculovirus. But they both have strong promoter activities.
3. ploh is a very late gene, so the product would be able to expression, even it is a cytotoxic .
4. It is a safe expression system to human and entironment.
Characteristic of BES-Silkworm:
5. Even the recombinant virus has a strong infecting activity on both the cells and the larva. So mass product might be expected .
6. Unlike prokaryotic expression system, product expressed by BES has a good way of after-processing.
7. Expression of fusion protein and polyprotein are permitted.
8. BES can express foreign genes from virus, bacteria, archaeon, fungi, plant or animal.
9. The cost of Bm-BES is very cheap.
hGM-CSF HCV-core antigen EGF: Gastricism IGF-I rabies virus: Mice were vaccinated orally (by bait),
gastrically (by stomach tube) with a recombinant virus expressing the rabies glycoprotein and nucleoprotein, IgAantibodies against rabies virus were detected at two weeks postvaccination. After challenge at one month postvaccination with CVS rabies virus, all mice that had developed antibodies were protected. It suggests that this recombinant virus might be a suitable vaccine for oral immunization against rabies.
10. The product could be taken orally: Edible
The Schematic protocol of Baculovirus-Silkworm Expression System
Silkworm rearing with artificial feedSilkworm rearing with artificial feed
Construction of Different FMDV Gene’s Combination for Expression in Silkworm
P12A3C
Expression of FMDV polypeptides in Expression of FMDV polypeptides in BmBm--N cells was N cells was analysedanalysed by IFATby IFAT
Bm-P12A3C BmBacPAK-6
Produced FMDV Virus Like Particles in Silkworm
Expression of FMDV polypeptides in silkworm larvae was estimated by the sandwich-ELISA
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
1/2048
1/4096
1/8192
1/16384
1/32768
1/65536
OD(492nm)
Expression of FMDV polypeptides in silkworm larvae was estimated by the sandwich-ELISA.
0.070
0.682
0.0540
1:16384
0.084
0.821
0.063
1:8192
0.087
0.995
0.085
1:4096
0.0630.0560.1500.1040.1130.1150.148
0.1710.241Negative control
1.1141.0831.2751.2841.3311.3261.245
1.3731.404Haemoly-mph with Bm-P12A3C
0.0830.1210.2270.3430.5560.7430.964
1.2041.315FMDV antigen
1:2048
1:1024
1:5121:2561:1281:641:321:161:8
Expression of FMDV polypeptides in silkworm larvae was estimated by the sandwich-ELISA
The expression time-course of FMDV polypeptides in Silkworm
The screening of high expression The screening of high expression viral clones viral clones
N OD(492nm)
N OD(492nm)
N OD(492nm)
N OD(492nm)
1 0.78 6 1.57 11 1.39 16 0.98
2 0.45 7 1.53 12 1.46 17 1.40
3 1.01 8 1.43 13 1.48 18 1.36
4 0.68 9 1.02 14 0.55 19 1.03
5 1.34 10 0.75 15 0.66 20 1.46
<8<8<8<8<8BmBacPAK-611
<8<8<8<8<8BmBacPAK-62
36036036045<8Bm-P12A3C50
90909020<8Bm-P12A3C45
45454520<8Bm-P12A3C122
90909020<8Bm-P12A3C124
36036018032<8Bm-P12A3C33
2821147-7number
dpvcLBPE-antibodyb
VaccineaAnimal #
FMDV-specific antibody response after vaccination with Bm-P12A3C’s
-4+mouth3Day 2BmBacPAK-611
-4+mouth3Day 2BmBacPAK-62
+---Bm-P12A3C50
-22Day 6Bm-P12A3C45
+---Bm-P12A3C122
+---Bm-P12A3C124
+---Bm-P12A3C33
ProtectioncLesion scoresb
Duration ofPyrexia(day)
Days of onset of pyrexiaa
vaccineAnimalnumber #
Protection and clinical signs in cattle after challenge with FMDV Asia I/HNK/CHA/05 strain
+1:45 1:901-15-1:451:20 1-14-1:81:8 1-13-1:451:20 1-12+1:451:20 1-11
1/9
+1:1801:1801-10+1:1801:1801-9-1:16 1:45 1-8+1:901:901-7
+++++
6.34
PD50
1/3
1:180 1:360 1-61:180 1:180 1-51:2561:90 1-41:45 1:32 1-31:128 1:180 1-2
Animalnumber
+
Protection
1:512 1:3601-1
1
LPBE-Antibody
(21)
LPBE-Antibody
(14)Immunize
dose
The result of PD50 test -1
-1:451:641-15-1:901:641-14-1:901:90 1-13+1:1801:1281-12+1:1801:1281-11
1/9
+1:1801:1281-10-1:1801:1281-9+1:1801:256 1-8+1:3601:2561-7
++++-
7.05
PD50
1/3
1:90 1:90 1-61:720 1:5121-51:901:45 1-41:3601:1801-31:512 1:64 1-2
Animal number +
Protection
1:512 1:3601-1
1
LPBE-Antibody
(21)
LPBE-Antibody
(14)Immunize
dose
The result of PD50 test-2
+1:901:902-15-1:451:452-14+1:1281:90 2-13+1:901:452-12-1:1801:902-11
1/9
+1:901:452-10+1:1801:902-9+1:901:45 2-8+1:3601:902-7
++++-
9.0
PD50
1/3
1:45 1:122-61:1801:2562-51:3601:5122-41:3601:3602-31:128 1:256 2-2
Animalnumber
+
Protection
1:1801:1802-1
1
LPBE-Antibody
(21)
LPBE-Antibody
(14)Immunize
dose
The result of PD50 test-3 15 cattlesvaccines from one larvasproduction
There are two different strains of Asia I FMDV prevalence in China, which are Asia 1/HNK/CHA/05 and JSL/06 isolate. When vaccinated with inactivated vaccine from Asia 1 /HNK/CHA/05, it can’t get the protection after challenge with JSL/06 strain and vice versa. So we designed an experiment to verify whether this produced antigen can get the protection against the two strains.All vaccinated cattle were challenged with 10,000 BID50 of AsiaI/HNK/CHA/05 at 21 dpv. The result showed the subunit vaccine potency could get 8.06、9.0、6.47 PD50. The vaccinated cattle were challenged with 10,000 BID50 of JSL/06 at 21 dpv. The result showed the subunit vaccine potency could get 6.47、5.20、6.47 PD50.
Cross-protection
ACKNOWLEDGEMENTS
Liu Jixing(Lanzhou VRI) Chen Yin Lin Xu’ai Yi Yongzhu He Jialu Shen Guifang Li Yinv Li Zhiyong and Yin Xiangping (Lanzhou
VRI) Zhang Zhidong and Li Yanmin (Institute for
Animal Health, Pirbright )
Thank you!