Preparation method of recombinant plasmid enhanced PCV2 vaccine immunoadjuvant of tibetan-pig IL-12 and application

A technology of IL-12 and Tibetan pigs, applied in the field of molecular immunology, can solve the problems of high cost, short timeliness, and short half-life

Active Publication Date: 2017-10-20
SICHUAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cytokines are naturally present in organisms and have a regulatory effect on immunity. In recent years, more and more attention has been paid to cytokines. However, cytokines have a short half-life in vivo, short timeliness and high cost. Therefore, cytokine genes are cloned into plasmids and transfected. Transfection to expression in vivo provides a way to solve this problem

Method used

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  • Preparation method of recombinant plasmid enhanced PCV2 vaccine immunoadjuvant of tibetan-pig IL-12 and application
  • Preparation method of recombinant plasmid enhanced PCV2 vaccine immunoadjuvant of tibetan-pig IL-12 and application
  • Preparation method of recombinant plasmid enhanced PCV2 vaccine immunoadjuvant of tibetan-pig IL-12 and application

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Experimental program
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Effect test

Embodiment 1

[0076] Embodiment 1, the construction of eukaryotic recombinant plasmid VRIL-12

[0077] 1. Extraction of RNA from peripheral blood lymphocytes

[0078] 1. Isolation and culture of Tibetan pig peripheral blood lymphocytes

[0079] (1) Collect 40 mL of blood from the anterior vena cava of Tibetan pigs, add 1640 culture medium and dilute in equal amounts;

[0080] (2) Add 5 mL of lymphocyte separation medium into a 15 mL centrifuge tube, then slowly add an equal amount of diluted blood, and centrifuge at room temperature for 30 min at 1500 r in a horizontal centrifuge;

[0081] (3) After centrifugation, four clear layers can be seen (from top to bottom: plasma layer, lymphocyte layer, separation liquid layer, red blood cell layer), carefully absorb the lymphocyte layer, a total of 2mL;

[0082] (4) Add 10 mL of 1640 medium for washing, and centrifuge at room temperature for 10 min at 1500 r;

[0083] (5) Discard the supernatant, add 10mL 1640 medium, mix by pipetting, and cen...

Embodiment 2

[0167] Example 2, the application of eukaryotic recombinant plasmid VRIL-12 as a vaccine adjuvant

[0168] 1. Preparation of vaccine adjuvants

[0169] 1. Preparation of chitosan nanoparticles (CS-IL-12) encapsulating VRIL-12

[0170] The VRIL-12 plasmid was encapsulated by the ion cross-linking method, and the nanoparticles were prepared as vaccine adjuvants. The specific method is:

[0171] (1) Preparation of the solution:

[0172] 10mg / ml TPP solution: Dissolve TPP in ddH2O to make the TPP concentration 10mg / ml to obtain a 10mg / ml TPP solution;

[0173] 2.4mg / ml chitosan solution: chitosan is dissolved in 1% (pH5.5) glacial acetic acid aqueous solution, so that the chitosan concentration is 2.4mg / ml, to obtain 2.4mg / ml chitosan solution;

[0174] Filter the prepared solution with a 0.22m microporous membrane to sterilize;

[0175] (2) Premixing of plasmid and TPP solution: Mix VRIL-12 solution (solvent is water) and appropriate amount of TPP solution evenly, and incuba...

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Abstract

The invention discloses tibetan-pig IL-12 and an application of recombinant plasmid thereof in preparing a PCV2 vaccine immunoadjuvant. The invention provides a protein which consists of an IL-12 protein P40 subunit, 2A, TPA and an IL-12 protein P35 subunit, wherein the amino acid sequence of 2A is the 324-344 site of a sequence 2, and the amino acid sequence of the TPA is the 345-369 site of the sequence 2. In the experiment, chitosan is used for wrapping IL-12 plasmid to prepare chitosan nano particles, and the chitosan nano particles and PCV vaccines are inoculated into experimental animals simultaneously, a mouse and a pig are respectively adopted as the experimental animals, and results prove that the IL-12 eukaryotic transfection plasmid can enhance immune response of the PCV vaccines effectively to provide an idea for developing a novel high-efficiency PVC immunoadjuvant.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to molecular immunology, and relates to the application of Tibetan pig IL-12 and its recombinant plasmid in preparing PCV2 vaccine immune adjuvant. Background technique [0002] Pork products are the main source of meat consumption for urban and rural residents and are closely related to people's lives. Since the 21st century, the annual number of live pigs and slaughtered pigs in my country has always accounted for about 50% of the world's total, ranking first in the world. While my country's pig industry has achieved world-renowned achievements, there are still many factors that restrict the healthy development of my country's pig industry, among which diseases are the most important factor. At present, there is still a large gap in the diagnosis and prevention of pig diseases in my country compared with developed countries in the world. Pig infectious diseases are still very serious...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/39A61K38/20A61P37/04A61P31/20A61P43/00
CPCA61K38/00A61K39/39A61K2039/552A61K2039/55538C07K14/5434C07K2319/02C07K2319/31
Inventor 高荣肖永乐方鹏飞宋婷玉邱文英陈祎李金海王泽洲吕学斌李江凌龚文波
Owner SICHUAN UNIV
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