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

Active Publication Date: 2019-02-05
HARBIN WEIKE BIOTECH DEV
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  • Description
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  • Application Information

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Problems solved by technology

On the other hand, the previous research of the present invention found that the E gene of phiX174 as a bacteriolytic gene can only last up to 4 hours. Previous studies have optimized the expression system. Although the bacteriolysis efficiency has been greatly improved, there are still bacteria Problems with short duration of lysis, resulting in low moult yields

Method used

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  • Application of pyroptosis-associated protein GSDMD (Gasdermin-D) for preparing bacterial ghost vaccine
  • Application of pyroptosis-associated protein GSDMD (Gasdermin-D) for preparing bacterial ghost vaccine
  • Application of pyroptosis-associated protein GSDMD (Gasdermin-D) for preparing bacterial ghost vaccine

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses application of pyroptosis-associated protein GSDMD (Gasdermin-D) for preparing a bacterial ghost vaccine. A bacteriolysis plasmid containing a pyroptosis-associated protein GSDMD coding gene is constructed and is converted into salmonella enteritidis; under the induction of gum sugar, bacteria are split to successfully obtain a salmonella novel bacterial ghost vaccine. An experiment proves that GSDMD mediated bacteriolysis has an extremely long persistent period, and a splitting rate is 99.9985% or more. After a novel bacterial ghost immune mouse prepared by the invention is adopted, an organism can be stimulated to generate powerful humoral immunity and cellular immunity; protective immunity response is induced; an experiment animal can be protected so as to resistsalmonella infection. The salmonella bacterial ghost vaccine prepared by the invention has a good immune protection effect.

Description

technical field [0001] The present invention relates to a bacteriolytic gene and its application in the preparation of vaccines, in particular to the application of the pyroptosis-related protein GSDMD in the preparation of slough vaccines. The application in vaccines, the invention belongs to the technical field of genetic engineering and immunology. Background technique [0002] Bacterial ghost is a bacterial shell without cytoplasm and nucleic acid. The bacteriolytic gene is expressed in bacteria, and the protein encoded by the gene can form a transmembrane pore structure in the bacterial shell, and under the action of osmotic pressure, the cytoplasmic content in the bacterium can be discharged through the pore, forming an empty bacterial shell. The slough has no toxic effect and is safe to use. It is a green vaccine with good protective effect. Sludge vaccines have adjuvant properties that allow them to enhance immune responses, such as T cell activation and mucosal im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K39/112A61P31/04C12N15/74C12N15/12
CPCA61K38/1729A61K39/0275A61K2039/523A61K2039/53A61P31/04C07K14/4723C12N15/74Y02A50/30
Inventor 于申业刘思国曹俊
Owner HARBIN WEIKE BIOTECH DEV
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