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Chimeric type DNA vaccine HSP70/CD80 for asthma prevention and immunotherapy

A DNA vaccine and immunotherapy technology, applied in the field of biomedicine, can solve the problems of insufficient safety and unsatisfactory efficacy.

Inactive Publication Date: 2013-06-19
钟森 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is that the efficacy of the existing pcDNA3.1-HSP70 / CD80 chimeric vaccine is not ideal enough and the safety is not good enough

Method used

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  • Chimeric type DNA vaccine HSP70/CD80 for asthma prevention and immunotherapy
  • Chimeric type DNA vaccine HSP70/CD80 for asthma prevention and immunotherapy
  • Chimeric type DNA vaccine HSP70/CD80 for asthma prevention and immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction and identification of the pVAXI-Hsp70 / CD80 vector of the present invention

[0035] (1) Materials and reagents:

[0036] pVAXI plasmid (invitrogen company), LIPOFECTAMINE 2000 liposome transfection reagent (invitrogen company), endonuclease Hind III, Xba I and T4 ligase (NEB company). PcDNA 3.1 - The Hsp70 / CD80 plasmid was constructed according to the description in the background art. Hsp70 monoclonal antibody (Lifespan Biosciences Company), CD80 monoclonal antibody (abcam Company), Western blot reagent (Shanghai Beyontian Biological Company).

[0037] (2) Construction process

[0038] PcDNA3--Hsp70 / CD80 plasmid and vector pVAXI were double-digested with endonucleases Hind III and Xba I, and the target gene Hsp70 / CD80 was connected to pVAXI with T4 ligase to construct pVAXI-Hsp70 / CD80. The constructed plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing verification.

[0039] (3) Identification of pVAXI-Hsp70 / CD80:

[...

Embodiment 2

[0041] Example 2 The present invention pVAXI-Hsp70 / CD80 expression and biodistribution detection

[0042] (1) Verification of eukaryotic expression

[0043] 4 μg (2 μl) of the pVAXI-Hsp70 / CD80 plasmid was encapsulated with 10 μl of LIPOFECTAMINE 2000 liposome transfection reagent and transfected into 293T cells. The cells were harvested at 24 hours, 48 ​​hours, and 72 hours, and the lysate was added to release the protein. The supernatant was mixed with 5x loading buffer and boiled to denature the protein. SDS-page electrophoresis, membrane transfer. Add Hsp70 and CD80 primary antibodies and incubate overnight at 4°C, and incubate with secondary antibodies for one hour after washing the membrane. After washing the membrane, there were single bands at 70kD (Hsp70) and 32KD (CD80) by chemiluminescence (results in figure 2 ), indicating that the vector can simultaneously express Hsp70 and CD80 proteins.

[0044] (2) In vivo expression and distribution detection

[0045] Exp...

Embodiment 3

[0058] Example 3 Study on the Immunoprotective Effect and Mechanism of pvAXI-Hsp70 / CD80 Chimeric DNA Vaccine on Asthma

[0059] (1) Test method.

[0060] 1. Experimental materials:

[0061] Rabbit anti-mouse PCNA (proliferating cell nuclear antigen) monoclonal antibody (mAb, Beijing Boaosen Company). Endotoxin-free plasmid purification system (Qiagen). IFN-γ, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-23, TGF-β, OVA-sIgE ELISA kit (ADL company), CD4 + CD25 + Regulatory T cell isolation kit (Miltenyi Biotec). SPF grade female BALB / c mice. Chicken ovalbumin (OVA, product of Sigma). 402A nebulizer (Jiangsu Yuyue Medical Equipment Co., Ltd.).

[0062] Preparation of DNA plasmids: Preparation of pVAXI-Hsp70 / CD80 and PcDNA with an endotoxin-free plasmid purification system 3.1 - Hsp70 / CD80 plasmid.

[0063] CD4 + CD25 + Regulatory T cell preparation: after the mice were anesthetized with ether, supercoiled plasmid DNA (5-iagen company) was prepared, dissolved in physiologi...

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a chimeric type DNA vaccine for asthma prevention and immunotherapy. The technical problem solved by the present invention is unsatisfied efficacy and poor safety of the existing pcDNA3.1-HSP70 / CD80 chimeric vaccine. The technical scheme of the present invention is to provide a new chimeric type DNA vaccine, which is pVAXI plasmid, wherein the pVAXI plasmid contains a HSP70 / CD80 chimeric protein encoding gene and can express HSP70 / CD80 chimeric protein in vivo. After the pVAXI vector is adopted to replace the pcDNA3.1(+) vector in the prior art, good effects are provided for asthma prevention and treatment, especially a strong Th1-type reaction can be induced, and IFN-gamma of the experiment subject can be significantly increased. In addition, the vector has Kanamycin resistance, does not cause any allergic reactions, and can provide a good asthma prevention and immunotherapy effect.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a chimeric DNA vaccine Hsp70 / CD80 for asthma prevention and immunotherapy. Background technique [0002] Bronchial asthma (asthma) is a chronic disease that seriously threatens public health worldwide. At present, there are about 300 million asthma patients in the world, and there are about 20 million asthma patients in my country. As the disease progresses, the lung function of asthmatic patients deteriorates progressively, making treatment more difficult and affecting the outcome of the disease. At present, it is believed that this is mainly due to the continuous damage and structural changes of the airway, that is, airway remodeling, which has attracted widespread attention. Epithelial gland hyperplasia and mucinous metaplasia lead to excessive mucus secretion, mucus plugs block the airway, and cause bronchial stenosis or even occlusion, as well as thickening of the basement membr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P11/06
CPCA61K39/39A61K2039/53A61P11/06
Inventor 钟森史小玲钟林
Owner 钟森
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