Avian interleukin-2 (IL-2) gene and eukaryon expressing plasmid and immunity reinforcing agent of bird vaccine

A technology of interleukin and expression plasmids, applied in the field of immune enhancers, can solve the problems of high price, incomplete control of infectious diseases, and limited range of use, so as to reduce the dosage, prolong immunity and duration, and enhance cell immunity. Immune Response and Effects of Humoral Immune Response

Inactive Publication Date: 2004-01-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to statistics, although about 10 billion chickens are vaccinated every year around the world, the occurrence of infectious diseases cannot be completely controlled.
The use of immune enhancers is an effective way to improve the immune effect of vaccines and control infectious diseases. However, the 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 Enhancers are imperative

Method used

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  • Avian interleukin-2 (IL-2) gene and eukaryon expressing plasmid and immunity reinforcing agent of bird vaccine
  • Avian interleukin-2 (IL-2) gene and eukaryon expressing plasmid and immunity reinforcing agent of bird vaccine
  • Avian interleukin-2 (IL-2) gene and eukaryon expressing plasmid and immunity reinforcing agent of bird vaccine

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Experimental program
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Embodiment 1

[0037] The present embodiment describes the method for obtaining chicken interleukin-2 (IL-2) gene provided by the present invention, comprising the following steps:

[0038] (1) Preparation of chicken spleen lymphocyte suspension Aseptically isolate the spleen of Xiaoshan chicken, a local breed in Zhejiang, grind it on a sterilized copper net (200 mesh) with a mortar, and place it in a Ca-free 2+ , Mg 2+ In PBS (pH7.2), centrifuge at 300g for 10min, take the supernatant and centrifuge at 500g for 30min to collect lymphocytes, wash with PBS three times, and then wash twice with RPMI 1640 culture medium. The living cells were counted by 1% trypan blue (1% Trypan blue) staining method, and after the number of living cells was more than 90%, it was diluted with RPMI 1640 culture medium to a certain concentration of single cell suspension.

[0039](2) Synthesis of chicken IL-2 cDNA by RT-PCR Cultured spleen lymphocytes according to the optimal conditions for in vitro induction of...

Embodiment 2

[0044] The present embodiment has described the obtaining method of chicken interleukin 2 (IL-2) gene eukaryotic expression plasmid pCI-IL-2, and its steps include:

[0045] (1) Amplification of the chicken IL-2 cDNA comprising the leader peptide sequence Primers were redesigned at both ends of the above-mentioned chicken IL-2 gene (432bp in full length, encoding a total of 143 amino acids including the leader peptide),

[0046] P3 (5'-CG GAATTC ATGATGTGCAAAGTACTGATC-3'),

[0047] P4(5'- GTC GAC TTA(His) 6 TTTTTGCAGATATCTCCACAAA-3'), the two ends of the primer were respectively introduced restriction sites EcoRI (underlined at P3) and SalI (underlined at P4), and considering the ease of purification of the expressed protein in the future, 6 His were designed in primer P4. Primers were synthesized in Shanghai Bioengineering Company. Using T-IL-2 as a template and P3 and P4 as primers, amplify the chicken IL-2 cDNA containing the leader peptide sequence according to the fol...

Embodiment 3

[0051] This embodiment has described the obtaining method of recombinant chicken interleukin 2 (IL-2) protein, comprises the following steps:

[0052] (1) Expression of Chicken Interleukin 2 (IL-2) Gene Eukaryotic Expression Plasmid pCI-IL-2 in Mammalian Vero Cells

[0053] The operation steps of liposome-mediated expression of chicken pCI-IL-2 in Vero cells are as follows:

[0054] Inoculate 2 × 10 on a 6-well cell culture plate 5 The number of cells in 2ml medium containing fetal bovine serum; 37 ℃ 5% CO2 incubator for 18-24h to 40-60% of the cell pool; prepare the following liquid in an Eppendorf tube: Solution A: Dilute 8μg DNA to 100μl Serum-free OPTI-MEMI Reduced Serum Medium medium (available from Gibco); Solution B: Dilute 10 μl Lipofectin Reagent (available from Life Technology) into 100 μl serum-free OPTI-MEM I Reduced Serum Medium medium middle. In order to achieve the best transfection effect, DNA should not exceed 20μg / ml and Lipofectin Reagent should not excee...

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Abstract

An interleukin-2(IL-2) gene of chicken is disclosed. An eukaryon expression plasmid containing said IL-2 gene is also disclosed, which uses pC as eukaryon expression vector. An immunopotentiator for fowls' vaccine is also disclosed, which is the aqueous solution of said eukaryon expression plasmid or the chicken's interleukin-2 protein expressed in eukaryon cell or animal body. Said immunopotentiator for fowls' DNA vaccines features high bioactivity, low cost and high effect to induce the immune response of cell and body fluid. It also elongate immune duration and reduce the dosage of vaccine.

Description

technical field [0001] The invention relates to a chicken interleukin-2 (IL-2) gene, a eukaryotic expression plasmid and an immune enhancer used as a poultry vaccine. Background technique [0002] Interleukin-2 (Interleukin-2, IL-2) is a kind of lymphokine produced by T lymphocytes, which can activate T cells, promote B cell differentiation and secrete antibodies, induce interferon gamma production, and enhance monocyte immunity. As well as the killing activity of NK cells, it plays a very important role in regulating the immune response of the body. In 1976, Morgan et al. first discovered mouse IL-2 in the culture supernatant of mouse lymphocytes. In 1983, Taniguchi et al. announced the successful cloning of the IL-2 gene. Subsequently, there were at least 30 kinds of IL-2 gene in mice, cattle, sheep, pigs, horses, etc. Mammalian IL-2 gene has been cloned successfully. However, in this regard, the research on poultry IL-2 represented by chickens i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/20A61P37/04C07H21/04C12N15/24C12N15/79
Inventor 于涟李建荣黄耀伟樊拥军
Owner ZHEJIANG UNIV
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