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Preparation of double promoter DNA anti-caries vaccine pCN-SSIE

A dual-promoter, anti-caries technology, applied in the field of biological vaccines, can solve the problem of lack of a good immune prevention method and the like

Inactive Publication Date: 2009-04-01
济南登益得科贸有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the particularity of the caries onset site and the special physiological environment and immune defense of the oral cavity, there has not been a good immune prevention method so far. top priority

Method used

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  • Preparation of double promoter DNA anti-caries vaccine pCN-SSIE
  • Preparation of double promoter DNA anti-caries vaccine pCN-SSIE
  • Preparation of double promoter DNA anti-caries vaccine pCN-SSIE

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Construction of plasmid pCN-SSIE

[0049] The construction process and specific steps of plasmid pCN-SSIE are as follows:

[0050] 1. PCR amplification of EGFP and SBR fragments

[0051] Using the plasmid pEGFP-N1 as a template, design primers to amplify the EGFP fragment from the plasmid pEGFP-N1 by PCR method, and add appropriate restriction sites at both ends of the fragment, and add the SalI site upstream, Added downstream is a NotI site. Primers were designed as follows:

[0052] Upstream 5'-GTCGACATGGTGAGCAAGGGCGA-3' with Sal I restriction site

[0053] Downstream 5'-GCGGCCGCTTACTTGTACAGCTCGT-3' with Not I restriction site

[0054] Finally, the gene encoding EGFP with restriction sites at both ends was obtained.

[0055] Using pcDNA3.1-SBR as a template, design primers to amplify the SBR fragment from pcDNA3.1-SBR by PCR, and add appropriate enzyme cutting sites at both ends of the fragment, and add Nco I upstream site, an EcoR I site was added...

Embodiment 2

[0070] Example 2: Preparation of anti-caries vaccine with plasmid pCN-SSIE

[0071] 1. Transform attenuated Salmonella SL3261 with plasmid pCN-SSIE, and select positive clones after LA screening and enzyme digestion identification.

[0072] 2.37 ℃ aeration enrichment culture for 16 hours, ventilation volume 25m 3 / h, so that the bacterial concentration and the copy number of the plasmid pCN-SSIE are suitable.

[0073] 3. Centrifuge the cultured bacterial solution at 4°C at 4000rpm for 10 minutes, discard the supernatant and resuspend the pellet with an appropriate volume of sterile PBS, centrifuge for 10 minutes under the same conditions, freeze and dry the pellet to obtain a freeze-dried vaccine.

[0074] The vaccine is available in oral enteric-coated capsules or powder; each serving contains 3 billion salmonella bacteria. The powder must be diluted with cold boiled water before use, and 300-500 ml of 5% baking soda is taken orally one hour before eating, and one dose is t...

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Abstract

The invention provides a method for constructing dual-promoter DNA anti-caries vaccine plasmid pCN-SSIE, and a preparation method of the anti-caries vaccine by the plasmid pCN-SSIE. Antigen gene SBR is inserted in the vector pCMVnir by the molecular cloning technology, green fluorescent protein reporter gene is added in the downstream of the SBR, and the SBR and the reporter gene pass through the interior of the ribosome and enter the site gene for connection so as to prevent the fusion expression thereof; and finally, a section of signal peptide sequence is fused at the 5' end of the SBR. The anti-caries vaccine improves the immunogenicity of the anti-caries vaccine, enhances the immune effect, has simple and convenient, effective and economical immune method with long duration, and can carry out immunity repeatedly with little side effects.

Description

technical field [0001] The invention utilizes modern molecular biology knowledge and genetic engineering technology to construct a novel anti-caries DNA vaccine plasmid pCN-SSIE, which is used for human caries prevention and treatment, and belongs to the technical field of biological vaccines. Background technique [0002] Dental caries is one of the most common diseases of all human beings, with a high incidence rate and great harm. It has been listed by the World Health Organization as the third major disease for prevention and treatment after coronary heart disease and cancer. Since the discovery of the close relationship between certain streptococci and caries in the 1960s, domestic and foreign scholars have done a lot of research on the etiology and pathogenesis of caries, and it has been shown that caries is a bacterial infectious disease. Streptococcus mutans is recognized as an important cariogenic bacteria. It plays an important role in the adhesion, aggregation an...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/31A61K48/00A61K39/09A61K9/48A61K9/14A61P1/02
Inventor 姜广水
Owner 济南登益得科贸有限公司
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