Toxin attenuation mutant for epsilon toxin of clostridium perfringens and application of toxin attenuation mutant

A technology of Clostridium perfringens and mutants, applied in the fields of application, bacteria, antibacterial drugs, etc., can solve the problems of easy side effects, incompleteness, and decreased immune effect of vaccinated animals, and achieve good immunogenicity and immunity protective effect

Active Publication Date: 2015-04-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently used commercial vaccines are mainly inactivated vaccines, which involve the inactivation of exotoxins in the preparation process, and there are potential safety hazards of toxin leakage or incomplete inactivation; in addition, various microtoxins in the culture supernatant and Bacterial metabolites often become allergens of immunized animals, and vaccinated animals are prone to side effects, and lead to decreased immune effect or even immune failure

Method used

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  • Toxin attenuation mutant for epsilon toxin of clostridium perfringens and application of toxin attenuation mutant
  • Toxin attenuation mutant for epsilon toxin of clostridium perfringens and application of toxin attenuation mutant
  • Toxin attenuation mutant for epsilon toxin of clostridium perfringens and application of toxin attenuation mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Expression and identification of recombinant Clostridium perfringens epsilon toxin attenuated body

[0050] 1. Experimental method

[0051] 1.1 Amplification of the target gene

[0052] According to the published epsilon toxin gene sequence, expression primers were designed for the epsilon protoxin gene Etx, and NcoI and XhoI restriction sites were introduced upstream and downstream of the expression primers, and mutation primers were designed for Y71A, F92A, Y169A and Y254A mutation sites point and the introduction of Y71E, Y71G, Y71S, Y71F and Y71W mutation sites. See Table 1 for primer sequences.

[0053] Table 1 is used to amplify the primers and sequences of Etx gene and its mutants

[0054]

[0055] Note: The italics are the introduced NcoI and XhoI restriction sites, and the underlines are the nucleotides encoding the mutated amino acids.

[0056] Using the genomic DNA of Clostridium perfringens type D reference strain CVCC C60-1 as a template, the...

Embodiment 2

[0095] Example 2 Detection of attenuation effect of ε toxin attenuated body in mouse model and immune protection test

[0096] 1. Experimental method

[0097] 1.1 Detection of the attenuation effect of the attenuated body in the mouse model

[0098] The virulence of the attenuated rEtx-Y71A screened in Example 1 in mice was detected, and the activated rEtx-Y71A was inoculated into mice through the intraperitoneal route, and 6 inoculation doses were set, which were 20ng, 50ng, 100ng, 1000ng, 10000ng and 100000ng, 5 mice were inoculated for each dose, and the recombinant wild-type mature toxin rEtx was set as a control, observed for 3 days after inoculation, and the side effects and death of the mice were recorded.

[0099] 1.2 Immune protection test of attenuated body in mouse model

[0100] Preparation of immunogen: The attenuated rEtx-Y71A protoxin screened in Example 1 was mixed with the oil adjuvant ISA 15A VG (SEPPIC, France) at a volume ratio of 85:15, fully mixed in a ...

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Abstract

The invention discloses a toxin attenuation mutant for epsilon toxin of clostridium perfringens and an application of the toxin attenuation mutant for the epsilon toxin of the clostridium perfringens. An epsilon toxin mutant for attenuating toxin in cells or animals is obtained by mutating tyrosine at a site 71 of mature toxin of the epsilon toxin of wide clostridium perfringens into non-aromatic amino acids. The invention further discloses a recombinant expression vector and a recombinant host cell which contain coding genes of the toxin attenuation mutant for the epsilon toxin of the clostridium perfringens. According to the recombined and expressed toxin attenuation mutant for the epsilon toxin of the clostridium perfringens, the complete toxin attenuation of mice in vitro and in vivo can be achieved, and good immunogenicity and immunizing protection are presented in a mouse model. The toxin attenuation mutant for the epsilon toxin of the clostridium perfringens and the coding genes of the toxin attenuation mutant can be used for preparing subunit vaccines of the epsilon toxin of the clostridium perfringens or subunit vaccines of multivalent clostridium toxin.

Description

technical field [0001] The present invention relates to Clostridium perfringens ε toxin, in particular to an attenuated mutant of Clostridium perfringens ε toxin, and the present invention further relates to the attenuated mutant of Clostridium perfringens ε toxin in the preparation of ε toxin sub The invention relates to an application in a unit vaccine or a multivalent Clostridium toxin subunit vaccine, and belongs to the field of attenuated mutants of Clostridium perfringens epsilon toxin and applications thereof. Background technique [0002] Clostridium perfringens (Clostridium perfringens) is a member of the genus Clostridium in the Bacillus family. This bacterium can produce at least 16 exotoxins and exert pathogenic effects through the toxins it produces, which can cause enterotoxemia in a variety of animals necrotic enteritis, traumatic gas gangrene in humans and animals, and food poisoning in humans. According to the types of four major lethal exotoxins α, β, ε, ι...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/31A61K39/08A61P31/04C12R1/145
CPCA61K39/00C07K14/33
Inventor 于力姜志刚常继涛
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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