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Anti-caries DNA (Deoxyribose Nucleic Acid) vaccine and preparation method and application thereof

A DNA vaccine and anti-caries technology, which is applied in pharmaceutical formulas, medical preparations with non-active ingredients, digestive system, etc., to achieve the effects of high safety, expanded application range, and easy promotion

Inactive Publication Date: 2013-04-03
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no literature report on the use of wapA in the preparation of DNA vaccines and its application in the study of caries prevention

Method used

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  • Anti-caries DNA (Deoxyribose Nucleic Acid) vaccine and preparation method and application thereof
  • Anti-caries DNA (Deoxyribose Nucleic Acid) vaccine and preparation method and application thereof
  • Anti-caries DNA (Deoxyribose Nucleic Acid) vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Construction of an anti-caries DNA vaccine——pVAX1-wapA

[0059] 1. Culture of Streptococcus mutans

[0060] The frozen Streptococcus mutans UA159 strain (gifted by the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, refer to the literature: Huang Zhengwei, Liu Zheng, Ma Rui, Tang Zisheng, Zhu Cailian, Optimization of electric shock transformation conditions for Streptococcus mutans, China Journal of Microecology, Issue 5, 2, 2007, page 404-405) Stretch culture and BHI plate medium (commercial plate medium, purchased from Shanghai Hengyuan Biotechnology Co., Ltd.), the culture condition is 37 ℃ anaerobic culture (80%N 2 10% CO 2 10%H 2 ). Pick the single clone grown on the plate and inoculate it into TY medium (1% tryptone 0.8% yeast extract pH5.0), and culture it with shaking at 37°C under airtight conditions. Bacteria were collected for subsequent experiments.

[0061] 2. Obtaining the genome of Streptococcus m...

Embodiment 2

[0125] Embodiment 2: the preparation of DNA vaccine chitosan nanocomposite

[0126] In order to improve the immunity of DNA vaccine, Na 2 SO 4 As a flocculant, DNA vaccine chitosan nanocomposites were prepared by self-assembly method.

[0127] The assembly steps are: chitosan (CS) is dissolved in NaAc buffer, with Na 2 S0 4 The DNA vaccine solutions were pre-heated to 50-55°C for 10 minutes, mixed quickly in equal volumes, vortexed for 1 minute, and left at room temperature for 30 minutes to obtain the DNA vaccine chitosan nanocomposite. The specific formulation conditions are shown in Table 6. Take 100 μl of nanoparticle suspension, make up 1ml with deionized water, and then use ZetasiserNano ZS laser particle size analyzer (Malvern, UK) to measure the average particle size, particle size distribution and Zeta potential of nanoparticles. The results showed that the grain size of the rice was uniform and the particle size distribution range was narrow. The results are show...

Embodiment 3

[0132] Embodiment 3: pVAX1-wapA and DNA vaccine chitosan nanocomposite eukaryotic expression ability testing process is as follows:

[0133] 1. 293A cell with 1×10 6 Spread 6-well plates at the density of cells / well and wait overnight for the cells to adhere to the wall.

[0134] 2. Transfect 293cells:

[0135] a, prepare pVAX1-wapA / cs, chitosan solution;

[0136] b. Gently mix pVAX1, pVAX1-wapA4μg and lipo10μl with 250μl serum-free medium respectively, and let stand for 5min;

[0137] c. Gently mix the serum-free medium mixed with plasmid and lipo, let stand at room temperature for 15-20min, and the total operation time shall not exceed 25min; Gently mix 500 μl serum-free medium;

[0138] d. Add the mixed solution into the 6-well plate, and change the serum-containing culture solution after 6 hours;

[0139] e. After 48 hours, the TRIzol method was used to extract total RNA. The steps are as follows: ①Add 1ml / well TRIzol directly to the 6-well cell culture plate to lyse ...

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Abstract

The invention belongs to the technical field of oral preventive medicine, and particularly provides an anti-caries DNA (Deoxyribose Nucleic Acid) vaccine. The anti-caries DNA vaccine is prepared by extracting the protein full-length gene of a wall-connected associated protein A (wapA) from a streptococcus mutans UA159 genome and cloning into a eukaryotic expression vector pVAX1. In order to enhance the immune capacity of the anti-caries DNA vaccine, the anti-caries DNA vaccine is prepared into a chitosan nanometer compound by taking chitosan as a vector. The invention also provides a preparation method and application of the anti-caries DNA vaccine. The anti-caries DNA vaccine provided by the invention has the advantages of high safety, natural antigen expression, lasting immune response and effective caries prevention.

Description

technical field [0001] The invention belongs to the technical field of oral preventive medicine, and in particular relates to an anti-caries DNA vaccine and its preparation method and application. Background technique [0002] The incidence of dental caries is second only to influenza, which seriously affects human health and quality of life. Due to my country's large population and limited medical resources, the incidence of caries is much higher than that in developed countries. It is particularly important to develop a caries vaccine that is easy to popularize to prevent and reduce the incidence of caries to protect national health. [0003] Streptococcus mutans (Streptococcus mutans hereinafter referred to as Streptococcus mutans) can closely adhere to the tooth surface and colonize the tooth surface, produce acidic metabolites, cause tooth demineralization and eventually lead to dental caries, and are the main cariogenic bacteria in humans. Glucosyltransferases (glucos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/09A61K47/36A61P1/02A61P31/04
Inventor 李洪娇陆一鸣向婧洁王少海鲁莹汪大林
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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