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Fowl vaccine immunopotentiator of chicken interleuken-18, and its application

A technology of interleukin and immune enhancer, applied in antiviral agents, peptide/protein components, medical preparations containing active ingredients, etc., can solve the problems of incomplete control of infectious diseases, frequent occurrence of immune failure, and limited use range and other issues, to achieve the effect of prolonging immunity, facilitating popularization and application, and enhancing resistance

Inactive Publication Date: 2006-11-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccination is currently the main means of preventing the occurrence of poultry infectious diseases, but this method cannot completely control the occurrence of certain infectious diseases, and intensive chicken farms often fail to immunize, causing huge economic losses to the poultry industry
The use of immune enhancers is an effective way to improve the immune effect of vaccines and control the occurrence of infectious diseases, but the currently widely used immune enhancers are artificially synthesized and expensive, which limits the scope of their use. Therefore, it is necessary to develop a cheap poultry immune enhancer agent imperative

Method used

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  • Fowl vaccine immunopotentiator of chicken interleuken-18, and its application
  • Fowl vaccine immunopotentiator of chicken interleuken-18, and its application
  • Fowl vaccine immunopotentiator of chicken interleuken-18, and its application

Examples

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Embodiment 1

[0027] This embodiment describes the method for obtaining chicken interleukin-18 (IL-18) gene provided by the present invention, comprising the following steps:

[0028] (1) Preparation of Chicken Spleen Lymphocyte Suspension Aseptically isolate the fresh spleen of 14-day-old Zhejiang breed Xiaoshan chick, grind it with a tissue grinder and add Ca-free 2+ , Mg 2+ Hank's solution, washed twice by centrifugation at 1300g for 20min, and then washed twice with RPMI1640 culture medium. After staining with trypan blue (1% Trypan blue) and counting viable cells, the cells were diluted into a single-cell suspension of a certain concentration with RPMI1640 (containing 10% calf serum, 1% penicillin and 1% streptomycin) culture medium.

[0029] (2) Synthesis of chicken IL-18cDNA by RT-PCR Culture spleen lymphocytes according to the optimal condition of IL-18 induced in vitro by screening, respectively stimulated by LPS with a final concentration of 10 μg / ml for 5h, 10h, 24h, and 48h to ...

Embodiment 2

[0037] The present embodiment has described the method for obtaining chicken interleukin 18 (IL-18) gene eukaryotic expression plasmid pCI-IL-18, and its steps include:

[0038] (1) Amplification of chicken IL-18 cDNA Primers were redesigned at both ends of the above-mentioned chicken IL-18 gene (591 bp in total, encoding 196 amino acids)

[0039] P3 (5'-CG GAATTC ATGAGCTGTGAAGAGATCG-3')

[0040] P4 (5'-CC GTC GAC TCATAGGTTGTGCCTTTCA-3')

[0041] Both ends of the primers were respectively introduced with enzyme cutting sites EcoR I (underlined at P3) and SalI (underlined at P4), and the primers were synthesized at Shanghai Boya Biotechnology Co., Ltd. Using T-IL-18 as a template and P3 and P4 as primers, the cDNA of chicken IL-18 was amplified according to the following PCR cycle program: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 45 s, annealing at 55°C for 1 min, extension at 72°C for 45 s, Extend at 72°C for 10 min, a total of 30 cycles. PCR product...

Embodiment 3

[0053] This example describes the method for obtaining recombinant chicken interleukin 18 (IL-18) protein. Include the following steps:

[0054] (1) Expression of Chicken Interleukin 18 (IL-18) Gene Eukaryotic Expression Plasmid pCI-IL-18 in Mammalian Vero Cells

[0055] The operation steps of liposome-mediated expression of chicken pCI-IL-18 in mammalian Vero cells are as follows:

[0056] Inoculate 2 × 10 on a 6-well cell culture plate 5 1 cell in 2ml medium containing fetal bovine serum; 37°C 5% CO 2 Cultivate in an incubator for 18-24h to 40-60% cell confluency: Prepare liquid in an Eppendorf tube: Slolution A: Dilute 8 μg DNA to 100 μl serum-free OPTI-MEM I Reduced Serum Medium medium (available from Gibco Company ); Solution B: Dilute 10 μl Lipofectin Reagent (available from Life Technology Company) into 100 μl serum-free OPTI-MEM IReduced Serum Medium medium. In order to achieve the best transfection effect, DNA should not exceed 20μg / ml and Lipofectin Reagent shoul...

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Abstract

A chicken's interleukin 18 as the immunopotentiator of fowls' vaccine is the aqueous solution of the eukaryon expression plasmid, which is a recombinant carrier composed of the p CI as the eukaryon expression carrier and interleukin 18 genes. It has high bioactivity and low cost.

Description

technical field [0001] The invention belongs to biological technology, and relates to an immune enhancer for poultry vaccines, in particular to chicken interleukin-18 immune enhancer for poultry vaccines and its application. Background technique [0002] Interleukin 18 (interleukin 18, IL-18) is a new type isolated and cloned for the first time in 1995 by Okamura et al. Cytokines, because this factor has a strong IFN-γ-inducing ability, it was originally named IFN-γ-inducing factor (IFN-γ-inducing factor, IGIF). Subsequent studies further found that IL-18 can not only induce IFN-γ, but also has a variety of biological functions, such as promoting the proliferation of Th1 cells, inducing the production of Th1 cytokines such as GM-CSF, IL-2 and TNF-α, And promote the differentiation and proliferation of T lymphocytes, enhance Fas-mediated Th1 cell cytotoxicity and NK cells, CTL cells and other biological functions, so IL-18 is a pleiotropic cytokine. In 1996, Usbio and other...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/20A61K39/39A61P31/20
Inventor 于涟许健李龙由振强万旺军
Owner ZHEJIANG UNIV
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