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Orally administrable immunostimulant product for aquaculture

An immune stimulation and product technology, applied in the field of orally administered immune stimulation products used in aquaculture, can solve the problems of inability to carry out strict dosage control, inactivation, and prevention of intestinal absorption of active antigens, so as to prevent fish diseases , to avoid the effect of side effects

Inactive Publication Date: 2011-10-05
PROBELTE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several disadvantages of the oral route are that tight dose control is not possible and large amounts of antigen are required for immunization
The biggest obstacle to oral administration is that antigens are often inactivated in the intestine by acidic environments or protease activity, and prevent intestinal absorption of active antigens

Method used

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  • Orally administrable immunostimulant product for aquaculture
  • Orally administrable immunostimulant product for aquaculture
  • Orally administrable immunostimulant product for aquaculture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Expression of gilthead seabream TNFα in Escherichia coli

[0049] Cloning of sbTNFα into pET15b

[0050] LPS-stimulated head kidney cDNA was used as template in PCR to use primers FE4 and RE5 ( figure 1 ) amplifies sbTNFα. Both primers contained a BamHI restriction site for subsequent cloning of the PCR product in the same site in plasmid pET15b (Novagen). 50 μM each dNTP, 0.2 mM primer, MgCl containing cDNA template 2 Amplification was performed in samples of 1X Buffer PLUS and 1 unit of Eco Taq PLUS DNA polymerase (Ecogen). Cyclic reactions were performed in an Eppendorf Mastercycler Gradient, including 1 cycle of 2 min at 95°C, 25 cycles of 45 s at 95°C, 45 s at 60°C, and 30 s at 72°C, followed by 1 cycle of 10 min at 72°C. The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen) and used 1 unit of T4 DNA ligase (New England BioLabs, Inc.) at a ratio of insert:plasmid=3:1 with plasmid pGEM-T at room temperature Easy (Promega) liga...

Embodiment 2

[0053] Example 2: Expression of gilthead seabream sbTNFα in Saccharomyces cerevisiae

[0054] Will His 6 -sb TNFα cloned into p424-GPD

[0055] Plasmid pET15b containing sbTNFα was used as template in PCR to use primers TNF-ECOF and TNF-ECOR ( figure 1 ) amplification of His including 613pb 6 - Fragment of sbTNFα. Both primers contained EcoRI restriction sites for cloning PCR products in the same site in plasmid p424-GPD (ATCC 87357). containing 5ng template, 2.5mM MgCl 2 , 50 μM of each dNTP, 0.2 mM primers, 1X High Speec Supplement, 1X Buffer PLUS, and 2 units of Eco Taq PLUS DNA polymerase (Ecogen) in samples. Cycling reactions were performed in Smart Cycler II (Cepheid), including 1 cycle of 95°C for 5 min, 30 cycles of 95°C for 30 s, 62°C for 45 s, and 72°C for 2 min, followed by 1 cycle of 72°C for 10 min cycle. PCR products were separated by electrophoresis in 0.8% agarose (Pronadisa) gels containing 0.5 μg / ml ethidium bromide and purified using the QIAquick Gel ...

Embodiment 3

[0059] Example 3: Expression of gilthead seabream sbTNF in Pichia pastoris

[0060] Will His 6 -sb TNFα cloned into pPICZA

[0061] Plasmid pET15b containing sbTNFα was used as template in PCR to use primers TNF-ECOPP and TNF-ECOR ( figure 1 ) amplification including His of 618pb 6- Fragment of sbTNFα. Both primers contained EcoRI restriction sites for cloning PCR products in the same sites in plasmid pPICZA (Invitrogen). containing 5ng template, 2.5mM MgCl 2 , 50 μm of each dNTP, 0.2 mM primers, 1X High Speec Supplement, 1X Buffer PLUS, and 2 units of Eco Taq PLUS DNA polymerase (Ecogen) in samples. Cycling reactions were performed in Smart Cycler II (Cepheid), including 1 cycle of 95°C for 5 min, 30 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 2 min, followed by 1 cycle of 72°C for 10 min cycle. PCR products were separated by electrophoresis in 0.8% agarose (Pronadisa) gels containing 0.5 μg / ml ethidium bromide and purified using the QIAquick Gel Extraction Ki...

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PUM

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Abstract

An orally administrable immunostimulant product comprises a microencapsulated cytokine and an enteric protection polymer to protect the cytokine, the cytokine is a fish, mollusc or crustacean cytokine, preferably a recombinant cytokine such as tumor necrosis factor a (TNFa) over-expressed in a host microorganism.

Description

Background of the Invention [0001] Aquaculture appears to be the only possibility to meet the growing demand for aqua-food that cannot be met by extractive fishing. Over the past 50 years, worldwide aquaculture production has increased from less than 1 million tonnes in the early 1950s to 59.4 million tonnes in 2004. This production level is valued at $70.3 billion. 69.6% of the world's total production was produced in China, 21.9% in Rest of Asia and the Pacific, 3.5% in Western Europe, and 0.4% in Central and Eastern Europe. In terms of the environment, aquatic production from mariculture (saltwater aquaculture) in 2004 was 30.2 million tonnes, accounting for 50.9% of the total production. Freshwater aquaculture provided 25.8 million tonnes (43.4%), while the remaining 3.4 million tonnes (5.7%) came from production from brackish environments. All data are from Fisheries Technical Paper 2006, State of World Aquaculture (FAO). [0002] Europe's aquaculture production accou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/50A61K39/39A61K38/19
CPCA61K38/191A23K1/004A23K1/188A61K38/1706A61K9/5026A23K1/1631A61K38/19A23K20/147A23K40/30A23K50/80A61P37/02A61P37/04
Inventor S·A·施特赖滕贝尔格M·佩尼亚尔韦尔梅利亚多J·A·洛佩斯马斯Y·佩德雷尼奥洛佩斯J·P·索拉冈萨雷斯P·马丁内斯奥尔蒂斯V·穆莱罗门德斯F·J·罗加索莱尔J·加林多比列加斯
Owner PROBELTE PHARMA
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