Application of pyroptosis-associated protein GSDMD (Gasdermin-D) for preparing bacterial ghost vaccine
A related protein and slough technology, applied in the fields of genetic engineering and immunology, can solve the problems of short duration of bacterial lysis and low slough yield, and achieve the goal of inducing protective immune responses, avoiding tissue damage, and controlling bacterial infection. Effect
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Embodiment 1
[0038] Embodiment 1: the construction of lysogenic plasmid
[0039] 1 Amplification of the target gene
[0040] The codon optimization of the GSDMD gene in GenBank was carried out for expression in Salmonella, and the nucleic acid fragment was synthesized by Suzhou Jinweizhi Company. Primers were designed as follows:
[0041] GSDMD-U(5'-CACGAATTCATGCCTAGCGCATTTG-3')
[0042] GSDMD-L (5'-AAAGTCGACATCACTCAGCAGGCTC-3')
[0043] EcoR I and Sal I restriction enzyme sites were introduced at the 5' ends of the upstream and downstream primers, respectively. Using the synthesized nucleic acid fragment as a template to amplify the GSDMD gene, its nucleotide sequence is shown in SEQ ID NO.1. The PCR amplification reaction system was 50 μL, including: 22 μL of deionized water, 25 μL of PrimeSTAR MAX Premix (2×); 1 μL of 10 μM upstream and downstream primers; 1 μL of nucleic acid fragment (containing 12.5 ng DNA). The cycle parameters of amplification are: 1) 94°C for 20s, 55°C for 10...
Embodiment 2
[0052] The preparation of embodiment 2 novel sloughs
[0053] 1 Electrotransformation and identification of lysogenic plasmid
[0054] In this embodiment, the Salmonella strain Sm6 is isolated from the sick chicken flock in this laboratory, and other Salmonella strains are also applicable to the present invention. The recombinant plasmid pSDiGc-GSDMD was electrotransformed into Salmonella Enteritidis SM6, and the specific operation steps were as follows: thaw the competent cells of Salmonella Enteritidis SM6 frozen in the refrigerator at -80°C on ice; add 1.5 μL of the plasmid to the competent cells, mix well Combine on ice for 10 minutes; transfer the mixture of plasmids and competent cells into a cold electroporation cup, and wipe off the water on the metal surface of the electroporation cup; set the electroporation parameters of the electroporation instrument to 2500V / cm, 200Ω, 25μF, 5.0ms; after electric shock , add LB medium preheated at 37°C to the electroporation cup, ...
Embodiment 3
[0060] The immune effect of embodiment 3 novel sloughs
[0061] 1 Preparation of the vaccine
[0062] Preparation of inactivated vaccines: Streak the Salmonella enteritidis SM6 frozen at -80°C on the Salmonella chromogenic medium, culture it upside down in a 37°C incubator for 12-18 hours, pick a single colony, and spread it on an LB plate, at 37°C Incubate the incubator upside down for 12-18 hours, wash the bacterial lawn with sterilized PBS, adjust the concentration, add 1.5‰ formaldehyde for inactivation, inactivate for 48 hours, and shake it from time to time, adjust the final concentration to 5×10 8 cfu mL -1 .
[0063] Preparation of inactivated vaccine plus adjuvant: take inactivated vaccine and mix equal volumes of Freund's adjuvant, the final concentration is 5×10 8 cfu mL -1 .
[0064] Novel slough vaccine: According to Example 2, the bacterial solution induced by adding arabinose was centrifuged at 6000r / min for 10min, and the supernatant was discarded. Wash w...
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