Clostridium perfringens beta toxin recombinant subunit vaccine and production method thereof
A technology of Clostridium perfringens and subunit vaccines, applied in the direction of microorganism-based methods, biochemical equipment and methods, vaccines, etc., can solve the problems such as difficulty in increasing the renaturation rate, and achieve immunogenicity and high-efficiency expression , the effect of avoiding biological safety hazards
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0064] ——Construction, expression and identification of Escherichia coli BLC2 strain (also known as Clostridium perfringens beta toxin 4 amino acid mutant in the present invention)
[0065] 1. Gene synthesis
[0066] According to the sequence of the natural protein gene of Clostridium perfringens β-toxin, the present invention designs 4 amino acid mutations after codon optimization, and the 212th arginine of the mature toxin of wild-type Clostridium perfringens β-toxin Acid is mutated to glutamic acid, leucine at position 268 is mutated to glycine, tyrosine at position 266 and tryptophan at position 275 are mutated to alanine, thus obtaining a beta toxin mutant that is non-toxic to animals . At the same time, MBP tag and 6×His tag were added to N-terminal and C-terminal respectively. The gene sequence GMBPCPB was synthesized by chemical synthesis m4 , containing a total of 2097 nucleotides.
[0067] The specific nucleic acid sequence is shown in SEQ ID No.1,
[0068]
...
Embodiment 2
[0092] ——Toxicity test of Clostridium perfringens beta toxin avirulent mutant to mice
[0093] To verify the actual attenuation effect of the mutant in vivo by measuring the toxicity of the Clostridium perfringens beta toxin avirulent mutant to mice. Purified Clostridium perfringens beta toxin 4 amino acid mutant recombinant protein mMBPCPB m4 , and the culture supernatant of Clostridium perfringens type C, inoculate 16-18g mice via tail vein at different doses, 5 mice per dose, 0.2mL / mouse. Results When the inoculation dose was 0.1 mg, all the mice were healthy and had no adverse reactions, while the wild-type control group could cause 5 / 5 mice to die when inoculated with 0.001 mL. This result indicated that the Clostridium perfringens beta toxin avirulent mutant was avirulent to mice and was identified as the toxin avirulent mutant.
[0094] Table 1 Recombinant protein mMBPCPB m4 Toxicity to mice
[0095]
Embodiment 3
[0097] ——Immunogenicity test of Clostridium perfringens beta toxin 4 amino acid mutant
[0098] 1. Bacterial culture
[0099] Inoculate the LB liquid medium containing kanamycin according to 2% of the total amount of the culture medium of the Escherichia coli genetic engineering strain BLC2 strain recombinantly expressing Clostridium perfringens beta toxin protein, and cultivate in a fermenter. The culture parameters were set as follows: culture temperature 37°C, pH value 7.0, dissolved oxygen higher than 20%. when culture OD 600 When the value is 10-15, lower the temperature to 15°C, and add IPTG with a final concentration of 0.5mmol / L to induce culture for 16 hours.
[0100] 2. Broken bacteria
[0101] Collect the bacteria by centrifugation, add 10mL of lysate (0.02mol / L Tris buffer (pH value 7.2), 0.3mol / L NaCl) to resuspend the bacteria according to the weight of each gram of the bacteria, and use a high-pressure homogenizer at 800bar The cells were crushed under press...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com