Preparation and application of pig interleukin-2 gene anti-infectious preparation

An interleukin, anti-infection technology, applied in gene therapy, anti-tumor drugs, introduction of foreign genetic material using vectors, etc., can solve the problems of short retention time, high cost, toxic and side effects, etc.

Inactive Publication Date: 2006-01-18
SICHUAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when IL2 is directly injected, due to its short retention time in the body, repeated administration of large doses is not only expensive, but also often accompanied by serious toxic and side effects. It has always been a hot application research field to study IL2 and other cytokines to improve the body's immune response

Method used

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  • Preparation and application of pig interleukin-2 gene anti-infectious preparation
  • Preparation and application of pig interleukin-2 gene anti-infectious preparation
  • Preparation and application of pig interleukin-2 gene anti-infectious preparation

Examples

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Embodiment Construction

[0070] 1. Cloning of Tibetan pig IL2 gene (TPIL-2 gene)

[0071] Collect 10 mL of blood from healthy Tibetan pig ear vein aseptically, separate Tibetan pig peripheral blood lymphocytes, add 10ug / ml Con A to stimulate, in CO 2 Incubate in an incubator at 37°C for 72 hours, extract total RNA from Tibetan pigs in time intervals (24, 48, 70 hours), and design a pair of Tibetan pig IL-2 gene cDNA amplification primers. The primer sequence is as follows:

[0072] Primer 1: CG GGATCC CAG TAA CCT CAA CTC CTG CCA CAAT,

[0073]Primer 2: GC CAA TTC CAA GTC AGT GTT GAG TAG ATG CTTT.

[0074] Using total RNA as template, add 10×RT-PCR reaction buffer 5μl, 25mM MgCl to the 50μl reaction system 2 10μl, 10mM dNTP5μl, RNase Inhibitor (40units / μl) 1μl, AMV RNase XL (5units / uL) 1μl, AMV-Optimized Taq (5units / uL) 1μl, 5'and 3'primers each 20uM, total RNA 1~2μl, water supplement To 50μl. The RT-PCR cycle parameters are: 50°C, 30min; 94°C, 2min, then 30 PCR cycles (94°C denaturation for 30s, 60°C an...

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Abstract

The invention discloses the preparation and application of porcine interleukin-2 gene anti-infectious preparation, which comprises, preparing plasmid chitosan nano particles with ion cross-linking method, dissolving porcine Interleukin-2 gene eucaryon plasmid (VPIL2) into tripolyphosphate solution and chitosan acetic acid solution with a molecular weight of 150000 and deacetylated degree of over 95%, water-bathing at 50 deg. C, keeping constant temperature for 20 minutes, homogeneously mixing plasmid solution and chitosan solution for 5 minutes by right proportion, obtaining VPIL2 plasmid chitosan nano particle liquid with a grain diameter of 40-60mn, Zeta electric potential of +25.6. The preparation has no cell toxicity and can resist the degradation to DNA by nuclease. The DNA nano granular formulation can be used for immunizing experimental animals separately through intramuscular injection or in combination with vaccines.

Description

Technical field [0001] 1. Biotechnology 2. Molecular Immunology [0002] Specifically, it includes the construction of porcine interleukin-2 gene eukaryotic expression plasmid, the molecular packaging of chitosan nanoparticles for Tibetan pig interleukin-2 gene eukaryotic expression plasmid and its application technology to enhance animal immune response and immune protection . Background technique [0003] Interleukin-2 (IL-2) is a key cytokine in the cytokine network, mainly secreted by helper T cells, NK cells and LAK cells; it can activate and promote the differentiation and maturation of T cells, NK cells and B cells It induces the activity of lymphokine-activated killer cells (LAK), and can also promote the synthesis and release of many lymphokines such as interferon and tumor necrosis factor, as well as the production of antibodies, and significantly enhance the body's immune function. IL-2 has been used to prevent and treat some diseases that are difficult to treat by con...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P37/04A61P35/00C12N15/63
Inventor 高荣武梅李惠王泽洲李江凌吕学斌吴凯源付满良杨毅王丽焕
Owner SICHUAN UNIV
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