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
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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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