Preparation method of recombinant panda IL-6 immunological adjuvant

A technology of IL-6 and immune adjuvant, which is applied in the field of preparation of recombinant giant panda IL-6 immune adjuvant, can solve the problems of unsatisfactory vaccines, poor safety, and low immune efficiency

Inactive Publication Date: 2013-11-27
四川卧龙国家级自然保护区管理局 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccine immunization is currently the main measure to prevent and control CDV, but the vaccines currently used are not very ideal, and there are two major problems of low immunization efficiency and poor safety

Method used

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  • Preparation method of recombinant panda IL-6 immunological adjuvant
  • Preparation method of recombinant panda IL-6 immunological adjuvant
  • Preparation method of recombinant panda IL-6 immunological adjuvant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Gene Cloning of Giant Panda IL-6

[0067] experimental method

[0068] 1.1 In vitro culture of giant panda peripheral blood lymphocytes

[0069] Under sterile conditions, take 2ml of healthy giant panda anticoagulant blood and place it in EDTA-Na 2 Vacuum tube, and then add an equal amount of D-Hanks solution to dilute. Slowly add 4ml of lymphocyte separation solution to the diluted giant panda peripheral blood, and centrifuge at 2000r / min for 20min. Carefully transfer the white blood cells to another test tube, add the same amount of RPMI1640 solution, centrifuge at 2000r / min for 15min, remove the supernatant, add 4ml of RPM11640 solution to the precipitate, wash twice, remove the supernatant; Suspend 10% calf serum, 100 μg / mL penicillin and 100 μg / mL streptomycin in RPMI1640 liquid, take 10 μL of cell suspension for counting, and dilute the cell suspension to 5×10 6 cells / mL, dispensed into cell culture flasks, CO 2 Incubate at 37°C for 24 hours in an incubator....

Embodiment 2

[0149] Prokaryotic Expression of Giant Panda Interleukin-6 and Preparation of Polyclonal Antibody

[0150] experimental method

[0151] 2.1 Primer design and synthesis

[0152] According to the sequencing results of the giant panda IL-6 gene and the analysis results of the signal peptide, the primers (IL-6P1 / P2) were designed using Oligo7.0 to amplify the cDNA base sequence encoding the mature peptide of IL-6. See the table below for specific primers (restriction sites are underlined).

[0153] Table 6 Primer Sequence

[0154]

[0155] 2.2 PCR amplification of the target fragment

[0156] Using the recombinant plasmid pMD18-T-IL6 as a template, a pair of synthetic specific primers was added for PCR amplification. The PCR reaction system is as follows:

[0157] Table 7

[0158]

[0159] The reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 40 s, annealing at 60°C for 40 s, and renaturation at 72°C for 50 s, and this step was per...

Embodiment 3

[0232] Adjuvant Effect of Recombinant Giant Panda Interleukin-6 on Genetically Engineered Canine Distemper Virus Vaccine in Mouse Model

[0233] Mouse canine distemper virus antibody (CDV-Ab) enzyme-linked immunoassay (ELISA), mouse interferon gamma (IFN-γ) ELISA kit, mouse interleukin 4 (IL-4) ELISA kit Purchased from Shanghai Times Biotechnology Co., Ltd., the rest of the reagents were of domestic analytical grade.

[0234] experimental method

[0235] 2.1 Preparation of cytokine adjuvant before immunization

[0236] After the purified recombinant cytokine protein was tested for biological activity, it was mixed with an equal volume of mineral oil, thoroughly mixed, and stored at 4°C for later use. Before immunization, mix equal volumes of recombinant cytokine protein and canine distemper genetically engineered vaccine, and mix thoroughly. The final concentration of the recombinant protein was based on the results of the MTT experiment, and the final concentration of IL-...

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Abstract

The invention discloses a preparation method of a recombinant panda IL-6 immunological adjuvant. The preparation method comprises the following steps of: cloning A1 panda IL-6 whole-genome; culturing A11 panda peripheral blood mononuclear cells in vitro; extracting an A12 total RNA (Ribonucleic Acid); synthesizing Al3cDNA; designing and synthesizing an A14 primer; carrying out PCR (Polymerase Chain Reaction) amplification on an A15 target gene; carrying out TA (trophoblast antigen) cloning on a PCR product of an A16 target gene; carrying out preliminary identification on A17 recombinant plasmids; and carrying out prokaryotic expression of A2 panda interleukin 6, and preparing a polyclonal antibody. The canine distemper is known as a destructive infectious disease and is the main fulminating infectious disease which threatens the population quantity and the life safety of pandas and seriously affects the healthy development of the pandas. The recombination panda IL-6 immunological adjuvant can be used for remarkably enhancing the immune effect of canine distemper vaccines and has important significance for the prevention and treatment of the canine distemper.

Description

technical field [0001] The present invention relates, in particular, to a preparation method of recombinant giant panda IL-6 (gpIL-6) immune adjuvant. Background technique [0002] The giant panda is known as China's "national treasure" and is a world-class rare wild animal. However, it is on the verge of extinction. In addition to the destruction of the giant panda's habitat due to human factors, disease and death have become one of the key reasons for the current endangered giant panda population. Among the more than 40 kinds of diseases that giant pandas suffer from, canine distemper IL-6 is the most severe infectious disease that threatens the population and life safety of giant pandas. Vaccine immunization is currently the main measure to prevent and control CDV, but the vaccines currently used are not very ideal, and there are two problems of low immunization efficiency and poor safety. Therefore, improving the immunity of giant pandas under safe dose vaccination has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70A61K39/175A61K39/39A61P31/14
Inventor 李德生朱玲张和民王承东易悦刘骁
Owner 四川卧龙国家级自然保护区管理局
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