Recombinant epsilon toxin and alpha toxin fusion protein vaccine of non-toxic clostridium perfringens and production method of fusion protein vaccine

A technology of Clostridium perfringens and fusion protein, which is applied in the direction of antibacterial drugs, bacterial antigen components, and resistance to vector-borne diseases, etc., to achieve the effect of preserving spatial conformation, maintaining immunogenicity, and reducing biosafety risks

Active Publication Date: 2019-05-14
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that the non-toxic region of CPA, CPA C Can play a good immune protection against natural CPA, but due to CPA C The molecular weight of the protein ...

Method used

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  • Recombinant epsilon toxin and alpha toxin fusion protein vaccine of non-toxic clostridium perfringens and production method of fusion protein vaccine
  • Recombinant epsilon toxin and alpha toxin fusion protein vaccine of non-toxic clostridium perfringens and production method of fusion protein vaccine
  • Recombinant epsilon toxin and alpha toxin fusion protein vaccine of non-toxic clostridium perfringens and production method of fusion protein vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] ——Construction, expression and identification of Escherichia coli BL13 strain

[0061] 1. Gene synthesis

[0062] The application is based on the natural gene sequences of Clostridium perfringens ETX and CPA, after codon optimization, the non-toxic region C-terminal (CPA C) were expressed in tandem, thereby obtaining a nontoxic toxin fusion protein for animals. At the same time, a 6×His tag was added to the C-terminus of the toxin fusion protein. The gene sequence GETX was synthesized by chemical synthesis m3 CPA C , containing a total of 1326 nucleotides. The specific nucleic acid sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0063] Sequence 1 (SEQ ID No.1):

[0064]

[0065] Sequence 2 (SEQ ID No.2):

[0066]

[0067]

[0068] 2. Construction of fusion expression vector

[0069] Synthetic GETX m3 CPA C As a template, the primer pair 1F / 1R (sequence 3 / sequence 4) is used for PCR amplification, wherein:

[0...

Embodiment 2

[0077] ——rETX m3 CPA C expression and identification of

[0078] 1. rETX m3 CPA C The expression will express rETX m3 CPA C The genetically engineered bacteria Escherichia coli (E.coli) BL13 strain was inoculated in 3 mL of LB liquid medium containing kanamycin, cultured with shaking at 37°C for 4 hours, and then added IPTG solution with a final concentration of 0.5M to induce culture for 4 hours . After the bacterial culture was completed, the bacterial cells were collected by centrifugation, and the bacterial cells were resuspended at a ratio of 10 mL of lysate [0.02 mol / L Tris buffer (pH 7.2), 0.3 mol / L NaCl] per gram of bacterial cells, and placed in an ice-water bath. The bacteria were lysed by ultrasonic for 30 min, and the crushing conditions were as follows: working for 9 s, resting for 9 s, and the ultrasonic power was 400 W. Centrifuge the lysed bacterial solution at 12000r / min for 30min at 4°C, discard the precipitate, and collect the supernatant. Take 30 μL...

Embodiment 3

[0081] ——rETX m3 CPA C purification of

[0082] Inoculate Escherichia coli (E.coli) BL13 strain in 1L LB liquid medium containing kanamycin for fermentation and culture, shake culture at 37°C OD 600 When it was 0.8, IPTG solution with a final concentration of 0.5M was added to induce culture for 4 hours. After the bacterial culture is completed, the bacterial cells are collected by centrifugation, and the bacterial cells are collected by centrifugation at 5000r / min for 5min, and the ratio of 10ml lysate (pH value 7.2 0.02mol / LTris buffer solution, 0.3mol / L NaCl) is added per gram of bacterial cell wet weight The bacteria were resuspended, and the bacteria were broken three times with a low-temperature high-pressure homogenizer at a pressure of 800 bar at 4°C. The lysate was centrifuged at 10,000 r / min at 4°C for 30 min, and the supernatant was collected. According to the instruction manual of the Ni-IDA affinity chromatography medium kit, rETX was soluble in the lysed super...

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Abstract

The invention relates to a recombinant epsilon toxin and alpha toxin fusion protein vaccine of non-toxic clostridium perfringens and a production method of the vaccine. According to an ETX mutant of non-toxic clostridium perfringens and C end fusion protein rETXm3CPAC of CPA, the non-toxic ETX mutant ETXm3 is in serial connection with a C end (CPAC) of CPA, soluble expression is achieved in escherichia coli BL21(DE3), the spatial conformation of natural toxin protein can be reserved to the highest extent, and accordingly the immunogenicity of escherichia coli can be maintained; the influence of a complex technology of inclusion body denaturation and renaturation on immunogenicity of the antigen protein is also avoided, the time of preparing the vaccine is shortened, and the production costis reduced. The C end of rETXm3CPAC contains six histidine (6*His) labels, and convenience is provided for protein purification; the obtained toxin fusion protein is completely free of toxicity in amouse body and has good safety, immunogenicity and immune protection performance in a rabbit model. The vaccine also has the advantages that the preparation technology is good, the efficacy of the vaccine is excellent, and A-type and D-type clostridium perfringens diseases are prevented at the same time.

Description

technical field [0001] The invention relates to a non-toxic Clostridium perfringens recombinant ε toxin and α toxin fusion protein vaccine and a production method thereof. It belongs to the field of veterinary biological products. Background technique [0002] Clostridium perfringeris is an important zoonotic bacterium that can cause traumatic gas gangrene and human food poisoning, as well as sudden gangrene in sheep, dysentery in lambs, necrotic enteritis in cattle and sheep, enterotoxin in cattle and sheep disease, which not only threatens human health, but also causes huge economic losses to animal husbandry. Because Clostridium perfringens disease has the characteristics of acute onset, short course of disease and high mortality rate, once the disease occurs, sudden death often occurs due to exotoxin poisoning before treatment, so immunization is an effective method to prevent and control the disease ( Lu Chengping. Veterinary Microbiology [M]. Beijing: China Agricultu...

Claims

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Application Information

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IPC IPC(8): A61K39/08A61K39/116A61P31/04
CPCY02A50/30
Inventor 刘莹陈小云杜吉革薛麒朱真王磊张莹辉印春生李启红姚文生康凯
Owner CHINA INST OF VETERINARY DRUG CONTROL
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