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Orally administrable immunostimulant product for aquaculture

An immunostimulation and aquaculture technology, applied in the field of orally administered immunostimulation products for aquaculture, can solve the problems of strict dosage control, inactivation, and prevention of intestinal absorption of active antigens, etc., to prevent fish Effects of disease, avoiding side effects

Inactive Publication Date: 2013-05-15
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 E. coli

[0049] Clone sbTNFα into pET15b

[0050] Use LPS-stimulated head kidney cDNA as a template in PCR to use primers FE4 and RE5 ( figure 1 ) Amplify sbTNFα. Both primers contained BamHI restriction sites for subsequent cloning of PCR products in the same site of plasmid pET15b (Novagen). It contains cDNA template, 50μM each dNTP, 0.2mM primer, MgCl 2 1X buffer PLUS and 1 unit of Eco Taq PLUS DNA polymerase (Ecogen) for amplification. The cyclic reaction was carried out in the Eppendorf Mastercycler Gradient, including 1 cycle of 95°C for 2 minutes, 25 cycles of 95°C for 45 seconds, 60°C for 45 seconds, and 72°C for 30 seconds, and a subsequent cycle of 72°C for 10 minutes. The PCR product was purified using QIAquick PCR Purification Kit (Qiagen), and 1 unit of T4 DNA ligase (New England BioLabs, Inc.) was used at room temperature to insert a fragment (insert): plasmid = 3:1 ratio with plasmid pGEM-T Easy (Promega) ...

Embodiment 2

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

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

[0055] Use plasmid pET15b containing sbTNFα as a template in PCR to use primers TNF-ECOF and TNF-ECOR ( figure 1 ) Amplification includes 613pb His 6 -Fragments of sbTNFα. Both primers contain EcoRI restriction sites, which are used to clone the PCR product in the same site of plasmid p424-GPD (ATCC 87357). It contains 5ng template, 2.5mM MgCl 2 , Each dNTP 50μM, 0.2mM primers, 1X High Speec additives, 1X buffer PLUS and 2 units Eco Taq PLUS DNA polymerase (Ecogen) sample for amplification. Carry out cyclic reactions in Smart Cycler II (Cepheid), including 1 cycle at 95°C for 5 minutes, 30 cycles at 95°C for 30 seconds, 62°C for 45 seconds, and 72°C for 2 minutes, followed by 1 cycle at 72°C for 10 minutes cycle. The PCR products were separated by electrophoresis in a 0.8% agarose (Pronadisa) gel containing 0.5 μg / ml ethidium bromide, and purif...

Embodiment 3

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

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

[0061] Use plasmid pET15b containing sbTNFα as a template in PCR to use primers TNF-ECOPP and TNF-ECOR ( figure 1 ) Amplification includes 618pb His 6 -Fragments of sbTNFα. Both primers contain EcoRI restriction sites, which are used to clone the PCR product in the same site of plasmid pPICZA (Invitrogen). It contains 5ng template, 2.5mM MgCl 2 , Each dNTP 50μm, 0.2mM primers, 1X High Speec additives, 1X buffer PLUS and 2 units of Eco Taq PLUS DNA polymerase (Ecogen) for amplification. Carry out cyclic reactions in Smart Cycler II (Cepheid), including 1 cycle at 95°C for 5 minutes, 30 cycles at 95°C for 30 seconds, 55°C for 45 seconds and 72°C for 2 minutes, and then 1 cycle at 72°C for 10 minutes cycle. The PCR products were separated by electrophoresis in a 0.8% agarose (Pronadisa) gel containing 0.5 μg / ml ethidium bromide, and purified using QIAquick Ge...

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Abstract

The present invention relates to an orally administrable immunostimulatory product comprising microencapsulated cytokines, which are cells of fish, molluscs or crustaceans, and an enteroprotective polymer for protecting said cytokines Factors, preferably recombinant cytokines, such as tumor necrosis factor alpha (TNFα) overexpressed in the host microorganism.

Description

Background of the invention [0001] Aquaculture seems to be the only possibility to meet the growing demand for aquatic food that cannot be met by extractive fishing. In the past 50 years, global aquaculture production has increased from less than 1 million tons in the early 1950s to 59.4 million tons in 2004. The value of this production level is $70.3 billion. 69.6% of the world’s total production is produced in China, 21.9% is produced in the rest of Asia and the Pacific, 3.5% is produced in Western Europe, and 0.4% is produced in Central and Eastern Europe. In terms of environment, the aquaculture production from mariculture (brine aquaculture) in 2004 was 30.2 million tons, accounting for 50.9% of the total production. Freshwater aquaculture provides 25.8 million tons (43.4%), while the remaining 3.4 million tons (5.7%) come from the production of brackish environments. All data are from FisheriesTechnical Paper 2006, State of World Aquaculture (FAO). [0002] Europe’s aqu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/19A61K39/39A61P37/04
CPCA61K9/5026A61K38/1706A61K38/191A23K20/147A23K40/30A23K50/80A61P37/02A61P37/04
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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