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Non-toxic tetanus toxin and clostridium novyi alpha toxine recombinant fusion protein

A technology of tetanus toxin and Clostridium novyi, which is applied in the direction of fusion with toxin, recombinant DNA technology, hybrid peptide, etc., can solve the problems of low yield, time-consuming, and incompleteness of Clostridium novyi toxin, and achieve high efficiency Expression and soluble expression, reduce biosafety risk, reduce the effect of production cost

Active Publication Date: 2019-07-23
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this type of vaccine has achieved certain effects in preventing Clostridium tetani and Clostridium novyi in animals, there are still some defects exposed in the use of these vaccines, for example, vaccine immunization is easy to cause local inflammation and toxicity in animals; The inactivation of exotoxin is involved in the preparation process, and there are biological safety hazards such as leakage of exotoxin or incomplete inactivation; in addition, the production of Clostridium novyi toxin is low, and it takes too long, and the influencing factors in fermentation excessive

Method used

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  • Non-toxic tetanus toxin and clostridium novyi alpha toxine recombinant fusion protein
  • Non-toxic tetanus toxin and clostridium novyi alpha toxine recombinant fusion protein
  • Non-toxic tetanus toxin and clostridium novyi alpha toxine recombinant fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] ——Construction, expression and identification of Escherichia coli BL / TN strain

[0071] 1. Gene synthesis

[0072] The application is based on the coding gene sequences of tetanus toxin (TT) and Clostridium novyi α toxin (Tncα), after codon optimization, the avirulent region C fragment (TTc) containing only tetanus toxin (sequence 1) and the N-terminal avirulent epitope of Tncα (3rd to 17th, 965th to 979th and 983rd to 997th amino acids) and the C-terminal (1800th to 2178th amino acids ) coding gene (SEQ ID NO: 2) was concatenated. At the same time, the 6×His amino acid tag coding sequence used for purification was added to the 3' end of the tandem gene. The gene sequence GTTc-Tcnα was synthesized by chemical synthesis nc (SEQ ID NO: 3), the amino acid sequence is as SEQ ID NO: 4.

[0073] 2. Construction of fusion protein expression vector

[0074] Synthetic GTTc-Tcnα nc As a template, the primer pair 1F / 1R (SEQ ID NO: 5 / SEQ ID NO: 6) was used for PCR amplificati...

Embodiment 2

[0102] ——rTTc-Tcnα nc expression and identification of

[0103] 1. rTTc-Tcnα nc expression

[0104] Inoculate Escherichia coli (E.coli) BL / TN strain in 4 mL of LB liquid medium containing kanamycin, place it at 37°C for shaking culture, when OD 600 When the temperature is 0.6-0.8, add IPTG solution with a final concentration of 0.5mM and place at 37°C and 15°C for induction and culture for 4h and 16h, respectively. 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 body weight, and placed in an ice-water bath. The bacteria were disrupted by ultrasonic for 15 minutes, the crushing conditions were: working for 9s, resting for 9s, and the ultrasonic power was 400W. The crushed bacterial liquid was centrifuged at 12000r / min for 10min at 4°C, and the supernatant was collected. Take 30 μL of ...

Embodiment 3

[0108] ——rTTc-Tcnα nc purification of

[0109] Inoculate the BL / TN strain of Escherichia coli in 1L LB liquid medium containing kanamycin for fermentation culture, shake culture at 37°C OD 600 When the temperature is 0.6-0.8, lower the temperature to 15°C, and add IPTG solution with a final concentration of 0.5mM to induce culture for 16h. After the bacterial culture is completed, collect the bacterial cells by centrifugation at 5000r / min for 5min, and resuspend the bacterial cells by adding 10ml of lysate (pH value 7.2 0.02mol / L Tris buffer, 0.3mol / L NaCl) per gram of bacterial cell wet weight Under the condition of 4°C, use a low-temperature high-pressure homogenizer to break the bacterial body three times at a pressure of 800 bar. The lysate was centrifuged at 10,000 r / min at 4°C for 30 min, and the supernatant was collected. According to the instructions of the Ni-IDA affinity chromatography medium kit, the soluble expressed rTTc-Tcnα in the cell lysate supernatant nc ...

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Abstract

The invention relates to non-toxic tetanus toxin and clostridium novyi alpha toxin recombinant fusion protein. The prepared recombinant fusion protein is produced in the manner that The prepared recombinant fusion protein is produced in the manner that through codon optimization, a tetanus toxin C fragment, C terminal of clostridium novyi alpha toxin, and N-end non-toxic epitope of clostridium novyi alpha toxin are subjected to fusing expression, so that the immunogenicity of two kinds of toxin protein can be reserved to the maximum extent, and biology potential safety hazard of natural toxincan be avoided. The recombinant fusion protein can be used for preparing clostridium tetani and clostridium novyi subunit vaccines. Compared with the clostridium tetani and clostridium novyi subunit vaccines commercialized in China at present, the non-toxic tetanus toxin and clostridium novyi alpha toxin recombinant fusion protein has the advantages of being simpler in preparation technology, lower in immunizing dosage, better in vaccine effects and the like, the biology security risk in the production process of the vaccine is greatly reduced, and the non-toxic tetanus toxin and clostridium novyi alpha toxin recombinant fusion protein is an ideal candidate vaccine antigen for upgrading and regenerating two clostridial toxin vaccines. When the non-toxic tetanus toxin and clostridium novyialpha toxin recombinant fusion protein and other antigens are in jointed preparation of a combined vaccine, the using dosage of the combined vaccine does not need to be increased, and the combined vaccine can be prepared.

Description

technical field [0001] The invention relates to a non-toxic tetanus toxin and Clostridium novyi alpha toxin recombinant fusion protein. It belongs to the field of biological products. Background technique [0002] Clostridium tetani and Clostridium novyi are anaerobic bacteria that can cause disease in humans and various animals, and are extremely harmful to human health and livestock and poultry breeding. secreted exotoxins. Among them, Clostridium tetani contains only one exotoxin, which is called Tetanus toxin (TT), while Clostridium novyi can mainly produce four exotoxins: α, β, γ, and δ. Clostridium novyi can be divided into three types: A, B and C according to the production of the above toxins. Among them, types A and B are the main types that cause disease in cattle and sheep, while type C is not virulent (Eeckhaut V, Boyen F, Pasmans F, et al. Clostridium novyi type B as a causative agent of bovine meat spoilage[J]. Anaerobe, 2012, 18(3):286-288.). Studies have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/08A61P31/04
CPCA61K39/08A61K2039/552A61K2039/70A61P31/04C07K14/33C07K2319/21C07K2319/55C12N15/62
Inventor 杜吉革刘莹张莹辉陈小云李旭妮李启红薛麒朱真王磊印春生姚文生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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