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
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  • 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.

Method used

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  • 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
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Example Embodiment

[0054] Example 1: Construction of pCN-SSIE plasmid, the steps are as follows:

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

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

[0057] I restriction site, add EcoR I restriction site 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 are digested with Sal I and Not I to obtain the target gene fragment EGFP and pIRES vector large fragment, and the two are connected in the ligation system to obtain pIRES-EGFP.

[0060] 3. The vector pCMVnir and the pIRES-EGFP obtained in step 2 were digested with restriction enzymes EcoRI...

Example Embodiment

[0065] Example 2: Construction of plasmid pCN-SSISG, the steps are as follows:

[0066]1. Use the plasmid pSG52 as the template to amplify the target gene fragment GBR by PCR, and add HindIII restriction sites in the upstream and XhoI restriction sites in the downstream; connect with the cloning vector pMD18-T for cloning and amplification .

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

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

Example Embodiment

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

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

[0079] 2. Incubate for 12 hours with aeration at 37°C, with a ventilation volume of 15m 3 / h to make the bacterial concentration and the copy number of plasmid pCN-SSISG reach the appropriate level.

[0080] 3. Centrifuge the cultured bacterial solution at 4°C at 4000 rpm 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, and freeze-vacuum dry the pellet to obtain a freeze-dried vaccine.

[0081] 4. The vaccine can be oral enteric-coated capsules or powder; the content of Salmonella per serving is 1 billion. The powder must be diluted with cold water before use. Take it 4 hours after a meal, take 200 ml of 5% baking soda by mouth half an hour befo...

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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

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Application Information

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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
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