Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation for pCN-SSISG of dual-promoter bivalent anti-caries DNA vaccine

A dual-promoter, anti-caries technology, applied in the field of biological vaccines, can solve the problem of inability to express antigens in prokaryotic cells

Inactive Publication Date: 2009-12-02
济南登益得科贸有限公司
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the vaccines known so far, the advantages of pCN-SSISG as a DNA anti-caries vaccine are mainly manifested in two aspects: 1. Most of the gene immunization vectors currently used are eukaryotic expression plasmids, which cannot express antigens in prokaryotic cells

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation for pCN-SSISG of dual-promoter bivalent anti-caries DNA vaccine
  • Preparation for pCN-SSISG of dual-promoter bivalent anti-caries DNA vaccine
  • Preparation for pCN-SSISG of dual-promoter bivalent anti-caries DNA vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: The construction of pCN-SSIE plasmid, the steps are as follows:

[0055] 1. Use the plasmid pEGFP-N1 as a template to amplify the target gene fragment EGFP by PCR, and add a SalI restriction site upstream and a NotI restriction site downstream.

[0056] Using the plasmid pcDNA3.1-SBR as a template, the target gene fragment SBR was amplified by PCR, and Nco was added upstream

[0057] I enzyme cutting site, EcoR I enzyme cutting site is added downstream.

[0058] The two gene fragments obtained above were respectively connected to the cloning vector pMD18-T to obtain plasmids pMD18-T-EGFP and pMD18-T-SBR for amplification.

[0059] 2. The plasmid pMD18-T-EGFP and plasmid pIRES amplified in step 1 were subjected to Sal I and Not I double enzyme digestion to obtain the target gene fragment EGFP and the large fragment of the pIRES vector, and the two were connected in the ligation system to obtain pIRES-EGFP.

[0060] 3. The vector pCMVnir and the pIRES-EGF...

Embodiment 2

[0065] Embodiment 2: The construction of plasmid pCN-SSISG, the steps are as follows:

[0066]1. Using the plasmid pSG52 as a template, use PCR to amplify the target gene fragment GBR, and add a HindIII restriction site in the upstream, and a XhoI restriction site in the downstream; after connecting with the cloning vector pMD18-T, perform clonal amplification .

[0067] 2. The plasmid pMD18-T-GBR and the vector pTriEx-4 amplified in step 1 were subjected to HindIII and Xho I double enzyme digestion to obtain the target gene fragment GBR and the large fragment of the pTriEx-4 vector, and the two were carried out in the ligation system Ligated to obtain plasmid pTriEx-4-GBR.

[0068] 3. Select the signal peptide sequence of tissue-type plasminogen (tPA) suitable for the extracellular secretion of GBR protein, and design the signal peptide sequence to eliminate the double sticky ends of EcoRI and HindI II enzyme cleavage sites: the upstream 5' end is formed by EcoRI enzyme clea...

Embodiment 3

[0077] Example 3: Preparation of anti-caries vaccine with plasmid pCN-SSISG

[0078] 1. Transform attenuated Salmonella SL3261 with plasmid pCN-SSISG, and select positive clones after LA screening and enzyme digestion identification.

[0079] 2.37 ℃ aeration enrichment culture for 12 hours, ventilation volume 15m 3 / h, so that the bacterial concentration and the copy number of the plasmid pCN-SSISG are suitable.

[0080] 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.

[0081] 4. The vaccine can be oral enteric-coated capsules or powders; the content of Salmonella in each serving is 1 billion. The powder must be diluted with cold boiled water before use. Take it after 4 hours after a meal, first take 200 milliliters of 5% baking soda orally ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides the construction of plasmid pCN-SSISG of bivalent dual-promoter DNA anti-caries vaccine and the preparation of anti-caries vaccine by plasmid pCN-SSISG. The construction method comprises the following steps: inserting an antigen gene SBR and an antigen gene GBR into a vector pCMVnir by using molecular cloning technology; connecting the antigen gene SBR and the antigen gene GBR through IRES to prevent fusion expression of the antigen gene SBR and the antigen gene GBR; and fusing a signal peptide gene on the 5' end of each antigen gene. The construction method increases the immunogenicity of the anti-caries vaccine and improves immunity effects. Moreover, the immunity method is simple, effective and economical, has long-lasting effect and can realize immunity repeatedly with insignificant side effects.

Description

technical field [0001] The invention uses molecular biology knowledge and genetic engineering technology to construct a novel anti-caries DNA vaccine plasmid pCN-SSISG, which is used for human caries prevention and treatment, and belongs to the technical field of biological vaccines. Background technique [0002] The etiology of caries has been clarified that caries is a bacterial infectious disease, and Streptococcus mutans is the main cariogenic bacteria, and its cariogenic effect depends on the adhesion, aggregation and plaque formation of bacteria on the tooth surface; and this The pathogenicity process mainly depends on two virulence factors of Streptococcus mutans, namely the surface protein Pac of Streptococcus mutans and glucosyltransferase GTF; the functional domains of the two have been clarified, respectively, the saliva binding of Pac region (SBR) and the glucan binding region (GBR) of GTF. Experiments have shown that the effective sealing of the above regions ca...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/63C12N15/54A61K48/00A61K39/00A61K9/48A61K9/14A61P1/02
Inventor 姜广水
Owner 济南登益得科贸有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com