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Viral myocarditis gene vaccine, its preparation method and application

A technology of viral myocarditis and gene vaccines, applied in the field of immunology, can solve the problems that naked DNA is difficult to retain, remove or degrade for a long time, and gene vaccines cannot effectively exert immune efficacy, etc.

Inactive Publication Date: 2004-10-13
上海欣安基因免疫与疫苗研究开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty of gene vaccines in inducing mucosal immune responses mainly lies in the fact that the naked DNA is difficult to persist in the mucosa and is easily cleared or degraded quickly, so that the gene vaccine cannot effectively exert its immune efficacy because it cannot effectively enter the local tissue

Method used

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  • Viral myocarditis gene vaccine, its preparation method and application
  • Viral myocarditis gene vaccine, its preparation method and application
  • Viral myocarditis gene vaccine, its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Construction of embodiment 1.pcDNA3-VP1

[0023] 1) First carry out the cultivation of Hela cells and the cultivation and passage of CVB3 virus, the method is as follows: the human cervical cancer cell line Hela cells are cultivated according to conventional methods, with 10% NBS, 2mM L-glutamine, 100U / ml penicillin and sulfuric acid Kanamycin-based RPMI-1640 medium was incubated at 37°C in 6% CO 2 Cultivate under the same conditions, subculture once every other day, gently digest the cells with 0.02% EDTA digestion solution at 37°C, and subculture at 1:2 to 1:4 after pipetting evenly. Infect approximately 5×10 6 For Hela cells, 80% of the cells were lysed by replicating virus after 40 hours of culture, and the liquid and cell fragments were centrifuged at 3000 rpm / min for 20 minutes, and the obtained supernatant was fresh CVB3 suspension.

[0024] 2) Obtain CVB3 VP1 gene from freshly cultured CVB3 by RT-PCR. The method is as follows: According to the CVB3 VP1 cDNA s...

Embodiment 2

[0029] Example 2. Preparation of novel viral myocarditis gene vaccine chitosan-pcDNA3-VP1

[0030] 1) Prepare chitosan solution and pcDNA3-VP1 solution respectively, the method is as follows: firstly prepare 0.02% chitosan solution with pH 5.7, weigh 0.02g chitosan (Sigma, MW=39000), and first mix with 500 μl ~ 1ml 1% HAc solution It dissolved slowly and was left at 37°C for 1 hr. Then weigh 0.042gNaAc, with 80ml H 2 O is dissolved, add the above-mentioned completely dissolved chitosan solution, mix well, and adjust the pH value to 5.7 with 1N NaOH, which is 0.02% chitosan solution with pH 5.7. PcDNA3 plasmid with 50mM Na 2 SO 4 Dissolve, the DNA concentration is 1mg / ml, and store at -20°C for later use.

[0031] 2) The chitosan-pcDNA3-VP1 complex was prepared by the complex co-precipitation method as follows: in a 55°C water bath, a 0.02% chitosan solution with a pH of 5.7 was dropped into the 80ug / ml plasmid DNA solution, and at the same time Shake for 20 seconds to for...

Embodiment 3

[0032] 3) Observe the freshly prepared chitosan-pcDNA3-VP1 complex with a transmission electron microscope, the method is as follows: drop the freshly prepared chitosan-pcDNA3-VP1 complex on a copper grid, room temperature for 5 minutes, negatively stain with uranyl acetate, and observe with a transmission electron microscope And take pictures. It was found that the freshly prepared solution contained spherical or elliptical particles with uniform shape, with a diameter of about 80-100nm ( figure 2 ). Example 3. In vitro expression of novel viral myocarditis gene vaccine chitosan-pcDNA3-VP1

[0033] Add 100ul of chitosan-DNA solution containing 1μg pcDNA3-VP1 DNA directly to 2×10 5 On the surface of Hela cells, add 2 μl Lipofectamine to 1 μg pcDNA3-VP1 DNA TM -2000 transfection complex was used as a control (1-3×10 5 The cells were seeded into 24-well plates, and 2ml of fresh antibiotic-free culture medium was replaced in the first 4 hours, and the cell density was prefer...

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Abstract

The present invention discloses a viral myocarditis gene vaccine, Said vaccine is constructed by using B3 type Coxsackie virus structural protein VPI gene and pcDNA carrier together, and its exterior is covered with a biological polysaccharide. It also discloses a method for preparing said gene vaccine and the application of said gene vaccine in preparation of medicine for preventing viral myocarditis.

Description

technical field [0001] The invention belongs to the field of immunology, and specifically relates to a novel gene vaccine which is jointly constructed by natural biological polysaccharide chitosan and the B3 type Coxsackie virus structural protein VP1 gene and can be used for the prevention and treatment of viral myocarditis and its preparation method . Background technique [0002] Mucosal immunity is one of the key directions of immunology and vaccine research in recent years. After many viruses and bacteria are infected from mucosal sites, the body fails to induce an effective mucosal immune response and the virus further spreads to the blood and cells throughout the body, making the specific CTL response induced by the body insufficient to remove a large number of diffused extracellular and intracellular Intravirus, leading to persistent infection such as virus. [0003] Viral myocarditis is mainly caused by coxsackie virus type B3 (CVB3) infection of mucosal infection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/125A61K48/00A61P31/14
Inventor 熊思东徐薇
Owner 上海欣安基因免疫与疫苗研究开发有限公司
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