Clostridium perfringen alpha toxin genetic engineering vaccine and application thereof

A Clostridium perfringens, genetic engineering vaccine technology, applied in the field of animal genetic engineering, can solve the problems of unbalanced vaccine response, different effects of antigenic components, induced diseases, etc., to prevent traumatic gas gangrene in animals, non-toxic Side effects, targeted effects

Inactive Publication Date: 2014-06-18
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These vaccines have a certain control effect on the epidemic of the disease, but they have the following disadvantages: the duration of the induced immune response is short, and multiple vaccinations are required; Different; due to the low level of induced immune response and the unbalanced vaccine response among the various antigenic components, it may induce disease; generally require the vaccine to be concentrated and purified; it cannot be vaccinated through natural routes, and it is not easy to...

Method used

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  • Clostridium perfringen alpha toxin genetic engineering vaccine and application thereof
  • Clostridium perfringen alpha toxin genetic engineering vaccine and application thereof
  • Clostridium perfringen alpha toxin genetic engineering vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Expression and purification of Clostridium perfringens alpha toxin protein

[0043] 1. Design primers

[0044] According to the α-toxin gene sequence submitted on Genebank (accession number: AY823400, its sequence is shown in Seq ID No: 1), amplify the partial specific gene fragment of α-toxin, design primers: primer1 and primer2, and add Nco I respectively and BamH I restriction site;

[0045] primer1: 5'-AGGGATGGAAAGATTGATGG-3' Nco I Seq ID No:3

[0046] primer2: 5'-TTATTTTATATTATAAGTTGAATTTCCTGA-3' BamH I Seq ID No:4

[0047] 2. Prepare the template

[0048] Pick up Clostridium perfringens type A strains with an inoculation loop and inoculate them on a blood plate medium (components: 100 ml of distilled water, 1 g of glucose and 3.8 g of bean agar powder, after sterilization, add 5 ml of sheep blood to pack onto six blood plates), anaerobic at 37°C (88%N 2 , 7%H 2 , 5%CO 2 ) for 36 hours. Take a single colony and inoculate it in a test tube contain...

Embodiment 2

[0099] Example 2: Alpha Toxin Protein Toxicity Assay

[0100] The culture supernatant of BL21(DE3)-α (i.e. supernatant 1), cells and cell lysates (the lysed bacterial liquid in step 5) and the culture supernatant of Clostridium perfringens virulent A528 Inoculate them on egg yolk agar plates respectively (Weigh 50g of the finished agar medium, add 1L of deionized water, stir and heat to boil until completely dissolved, divide into Erlenmeyer flasks, sterilize at 121°C for 15min, cool to about 50°C, add Suspend 10-15mL of 50% sterile egg yolk saline, shake well and immediately pour into six plates, solidify), and anaerobically culture at 36°C for 24 hours, the culture supernatant, cells and cell lysate of BL21(DE3)-α are all There is no characteristic turbid ring, but a milky white turbid ring appears in the culture supernatant of Clostridium perfringens virulent A528, indicating that BL21(DE3)-α no longer has the phospholipase C activity of α toxin; intraperitoneal injection o...

Embodiment 3

[0102] Example 3: Safety inspection of α-toxin genetically engineered vaccine

[0103] 4 rabbits with a body weight of 1.5-2 kg were injected with 5 ml of the vaccine intramuscularly, and observed for 10 days, all of them were healthy and alive, and no necrosis occurred at the injection site.

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Abstract

The invention relates to a clostridium perfringen alpha toxin genetic engineering vaccine and application thereof. The clostridium perfringen alpha toxin genetic engineering vaccine is prepared by designing a specific primer, implementing polymerase chain reaction (PCR) amplification on A-type clostridium perfringen DNA to obtain an alpha toxin gene segment, cloning the gene segment onto a pMD18-T Vector, selecting a positive recon and linking the positive recon onto a PET-28 alpha (+) plasmid; introducing the recombinant plasmid to a receptor strain to successfully construct a recombinant strain BL21 (DE3)-alpha; emulsifying the prepared alpha toxin protein and an aluminum hydroxide adjuvant at a volume ratio of 9:1, and adding 0.01% thiomersal to obtain the clostridium perfringen alpha toxin genetic engineering vaccine. The clostridium perfringen alpha toxin genetic engineering vaccine is easy for realization of industrial production, simple to operate and good in safety, and is capable of effectively preventing such diseases as animal necrotic enteritis, enterotoxemia, and human and animal traumatic gas gangrene caused by the A-type clostridium perfringens.

Description

(1) Technical field [0001] The invention discloses a Clostridium perfringens alpha toxin genetic engineering vaccine and its application, belonging to the technical field of animal genetic engineering. (2) Background of the invention [0002] Clostridium perfringens is one of the main pathogenic bacteria that cause various animal necrotic enteritis, enterotoxemia, human and animal traumatic gas gangrene and human food poisoning. The pathogenic factor of this bacteria is the exotoxin it produces. So far, there are 12 exotoxins (α, β, γ, δ, ε, η, θ, ι, κ, λ, μ, and ν) of Clostridium perfringens that have been found, but they play the main pathogenic role. There are only 4 species (α, β, ε and ι), according to which the bacteria are divided into 5 toxin types such as A, B, C, D and E. All types of Clostridium perfringens produce α-toxin, which is the main virulence factor of Clostridium perfringens. It has two enzyme activities, phospholipase C and sphingomyelinase, and can si...

Claims

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Application Information

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IPC IPC(8): A61K39/08C12N15/70C07K14/33A61P31/04C12R1/145C12R1/19
Inventor 柴同杰王叶
Owner SHANDONG AGRICULTURAL UNIVERSITY
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