A Recombinant Fusion Protein of Avirulent Tetanus Toxin and Clostridium perfringens β Toxin

A technology of Clostridium perfringens and tetanus toxin, which is applied in the direction of recombinant DNA technology, bacteria, hybrid peptides, etc., can solve the problems of difficulty in increasing the annealing rate, reduce production costs, and avoid immunogenicity of antigenic proteins The impact and the effect of reducing biosafety risks

Active Publication Date: 2020-12-25
CHINA INST OF VETERINARY DRUG CONTROL
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the expression products in inclusion bodies do not have biological activity, denaturation and renaturation treatment is required, and protein denaturation and renaturation is an extremely complicated process. The renaturation conditions of different proteins are different, and the renaturation rate It is often difficult to improve, which is the main constraint limiting its application

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A Recombinant Fusion Protein of Avirulent Tetanus Toxin and Clostridium perfringens β Toxin
  • A Recombinant Fusion Protein of Avirulent Tetanus Toxin and Clostridium perfringens β Toxin
  • A Recombinant Fusion Protein of Avirulent Tetanus Toxin and Clostridium perfringens β Toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] ——Construction, expression and identification of Escherichia coli BL / TB strain

[0068] 1. Gene synthesis

[0069] The application is based on the coding gene sequences of tetanus toxin (TT) (sequence 1) and Clostridium perfringens type C CPB (sequence 2), after codon optimization, the non-toxic tetanus toxin containing only tetanus toxin Region C fragment (TTc) and four amino acid mutations (arginine at position 212 to glutamic acid, leucine at position 268 to glycine, tyrosine at position 266 and tryptophan at position 275 The CPB-encoding gene for alanine) was concatenated. At the same time, the coding sequence of the 6×His tag used for purification was added to the 3' end of the tandem gene. The gene sequence GTTc-CPB was synthesized by chemical synthesis m4 (Sequence 3). The specific nucleic acid sequence is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No.4.

[0070] 2. Construction of recombinant fusion protein expression vector

[00...

Embodiment 2

[0094] ——rTTc-CPB m4 expression and identification of

[0095] 1. rTTc-CPB m4 SDS-PAGE identification of expression

[0096]Inoculate Escherichia coli (E.coli) BL / TB strains in 4 mL of LB liquid medium containing kanamycin, place them in shaking culture at 37°C, and 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 ...

Embodiment 3

[0100] ——rTTc-CPB m4 purification of

[0101] Inoculate the BL / TB strain of Escherichia coli in 1L LB liquid medium containing kanamycin for fermentation and culture, and shake at 37°C for 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 instruction manual of the Ni-IDA affinity chromatography medium kit, the soluble expressed rTTc-CPB in the cell lysate supernat...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention relates to non-toxic tetanus toxin and clostridium perfringens beta toxin recombined fusion protein. By means of codon optimization, clostridium tetani fragments C and the recombined fusion protein containing multiple clostridium perfringens beta toxin mutants with amino acid mutation, the immunogenicity of two kinds of toxin protein is reserved to the maximum extent, and potential biosafety hazards caused by single amino acid mutation are avoided. The recombined fusion protein can be used for preparing unit vaccines of clostridium tetani, B type clostridium perfringens and C type clostridium perfringens; compared with a current commercial clostridium natural toxin inactivated vaccine in China, the recombined fusion protein has the advantages of being simple in preparation process, low in immunizing dose, excellent in vaccine potency and the like, the biosafety risk in the vaccine production process are greatly reduced, and the recombined fusion protein is an ideal candidate vaccine antigen for upgrading and updating the three kinds of clostridium toxin vaccines. When the recombined fusion protein is used for preparing a combined vaccine with other antigens, the combined vaccine can be prepared without increasing the use dose of the combined vaccine.

Description

technical field [0001] The invention relates to a recombinant fusion protein of avirulent tetanus toxin and Clostridium perfringens beta toxin. It belongs to the field of biological products. Background technique [0002] Clostridium tetani and Clostridium perfringens are anaerobic bacteria that can cause disease in humans and various animals, and are extremely harmful to human health and livestock and poultry breeding. The pathogenic factors of these two types of Clostridium Both are exotoxins secreted by bacteria. Clostridium tetani contains only one exotoxin, which is called Tetanus toxin (TT), while Clostridium perfringens has at least 20 exotoxins, but in the form of α (CPA), β (CPB) , ε (ETX) and ι (CPI) are the most important exotoxins, and accordingly the bacteria can be divided into 5 toxin types A, B, C, D, E (Revitt-Mills, S; Rood, J ; Adams, V. Clostridium perfringens extracellular toxins and enzymes: 20 and counting. Microbiol. Aust. 2015, 36, 114–117.). Bec...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/116A61K39/114A61P31/04C12R1/19
Inventor 杜吉革刘莹王磊陈小云李旭妮李启红朱真薛麒张莹辉姚文生印春生
Owner CHINA INST OF VETERINARY DRUG CONTROL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap