44 results about "Clostridium perfringens toxoid" patented technology

The invention discloses a preparation method and application of veterinary-use A type clostridium perfringens toxoids. The preparation method of the toxoids includes the following steps that A type clostridium perfringens production strains are inoculated into a culture medium for fermentation and culture, a fermentation product is obtained, then the fermentation product is inactivated and detoxified in L-lysine and a formaldehyde aqueous solution under the pH of 6.8, and centrifugal supernatant of the successfully inactivated and detoxified qualified bacterial solution refer to toxoids. The culture medium includes casein peptone, yeast extract powder, CaCl2, ZnSO4 7H2O, Na2HPO4 12H2O, KH2PO4, and glucose. The toxicity of the A type clostridium perfringens toxoids prepared by the method can be raised to be 12.5 times of the standard for seedling cultivation, the neutralization titer, in first and second immune serum of domestic rabbits, of the toxoids can be raised to 4 and 50 times ofa traditional process respectively and maximally, and the ratio of the serum titer to cost can be raised to 48.8 and 1216.4 times of the traditional process.

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.

The invention relates to a clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method. According to the clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method, alpha toxin protein obtained via prokaryotic expression is taken as an immunogen; monoclonal antibodies obtained via hybridoma technique are taken as detection antibodies and capture antibodies; reaction conditions are optimized via experiments; alpha toxin protein samples with a series of concentration are used for construction of a standard curve; the double-antibody sandwich ELIS method is established; and indexes of the double-antibody sandwich ELIS are verified. The clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method specifically comprises following steps: (1) prokaryotic expression of alpha toxin; (2) preparation of anti-alpha toxin monoclonal antibodies; (3) establishment of the double-antibody sandwich ELIS method; (4) establishment of the standard curve; and (5) performance evaluation on the double-antibody sandwich ELIS method. The clostridium perfringens alpha toxin double-antibody sandwich ELIS quantitative determination method is excellent in specificity, and high in sensitivity and stability, is fast and convenient, and can be used for effective quantitative determination of alpha toxin in A-E type clostridium perfringens cultural supernatants; and the high-efficient detection method is provided for alpha toxin determination.

The invention discloses a preparation method and a special culture medium of veterinary D-type clostridium perfringen toxin. The culture medium per 100ml comprises the following components: 1-1.5g soy peptone, 1-1.5g casein peptone, 0.5-0.75g yeast extract, 0.5-0.75g Na2HPO4.12H2O, 1-1.5g dextrin and the balance of water, wherein a pH (potential of hydrogen) value of the culture medium is 8.0-8.5. The preparation method of the D-type clostridium perfringen toxin comprises the steps of inoculating a D-type clostridium perfringen production strain into the culture medium, collecting a culture material, performing centrifugation, and then filtering a supernate. With the adoption of the method, the maximum toxicity can be improved to be 45 times a seedling standard in Regulations on National Veterinary Biological Products, and an output-input ratio can be increased to be 30-225 times that of the original traditional technology. In addition, corresponding serum neutralization titer of a toxoid vaccine prepared by the D-type clostridium perfringen toxin on a rabbit and a sheep is also improved to be 8.3 and 13.3 times a regulation standard respectively.

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.

The invention discloses a type B Clostridium perfringens exotoxin, a preparation method thereof, a toxin-producing medium and an application thereof. Wherein every 1000ml described toxin-producing medium is made of the raw materials of following weight ratio: peptone 15-25g, yeast extract powder 3-5g, NaCl 3-4g, NaCl 2 HPO 4 12H 2 O 5‑10g, dextrin 5‑10g or crude dextrin 5‑10g, and the rest is water for injection. The virulence of the type B Clostridium perfringens toxin prepared by the method of the invention can reach up to 1 mouse MLD≤0.0006mL, which is 3.3 times higher than the seedling production standard. Moreover, the neutralization titer of the corresponding serum neutralization titers of rabbits and sheep in the triple inactivated vaccine of sheep rapid epidemic disease and sudden attack (or lamb dysentery) enterotoxemia also reaches or exceeds the standard of relevant regulations.

The invention discloses a humanized single-chain antibody 7D of Clostridium perfringens α toxin, which is screened from the phage antibody library Source bioscience, and is a fully human anti-Clostridium perfringens α toxin antibody, which can be realized Neutralizing the therapeutic effect of toxins, while overcoming various side effects of heterologous antibodies, eliminating the cumbersome steps and high cost of humanized transformation of heterologous antibodies, due to the small molecular weight of single-chain antibodies and strong penetrating ability in vivo, It quickly reaches damaged tissues and cells to play an anti-toxin effect, thus achieving the dual purpose of economy and high efficiency. The α-toxin has certain homology with other types of toxins of Clostridium perfringens, and the antibody drug may have inhibitory and therapeutic effects on other types of toxins, and can also be used as a detection reagent to detect α-toxins of Clostridium perfringens.

The invention discloses a preparation method of an E-type clostridium perfringens toxin vaccine. The method comprises the following steps: 1) an E-type clostridium perfringens bacterial strain is inoculated to an aseptic blood dish medium, anaerobic cultivation is carried out, and recovery of the bacterial strain is performed; 2) the recovered E-type clostridium perfringens bacterial strain is inoculated to a liquid thioglycollate fluid medium, enrichment culture is carried out under anaerobic condition, and seed liquid is prepared; 3) the seed liquid is inoculated to a toxin-producing medium,anaerobic cultivation is carried out, and exotoxin liquid is prepared; and 4) the prepared exotoxin liquid is subjected to inactivation and emulsification, and merthiolate is added to obtain the E-type clostridium perfringens toxin vaccine. The E-type clostridium perfringens toxin vaccine has the advantages of strong immunity, and no toxic and side effect. The vaccine can effectively prevent diseases such as calf and lamb dysentery due to the strain.

This invention pertains in part to the development of a vaccine for poultry against necrotic enteritis (NE). The vaccine utilizes a protective antigen that is a mutated, full-length, non-toxic Clostridium perfringens (Cp) α-toxin protein (Mcpa). Utility of this vaccine was demonstrated by reduction of lesion severity in NE challenge trails, for example. Also disclosed herein are novel approaches for producing this vaccine in significant quantities. One exemplified approach involves producing NE vaccine (mutated alpha toxin) in bacterial expression systems, preferably utilizing the Pseudomonas fluorescens system, for commercial use in controlling NE in the poultry industry. The subject vaccines can be administered preferably to chickens in several different ways. A novel, Type VI alpha toxin from chicken isolates of Cp is also disclosed.

The invention relates to a Clostridium perfringens epsilon toxin recombinant subunit vaccine and a production method thereof. The Clostridium perfringens epsilon toxin recombinant subunit vaccine prepared by the present invention is produced by codon-optimized recombinant Clostridium perfringens epsilon toxin protein containing 3 amino acid mutations, that is, the natural toxin protein is retained to the greatest extent Integrity and spatial conformation, thereby maintaining its immunogenicity, and avoiding the biological safety hazards caused by single amino acid mutations. The vaccine also has the advantages of simple preparation process, low immune dose, and much higher vaccine efficacy than existing vaccines. Compared with the current commercialized Clostridium perfringens natural toxin inactivated vaccine in China, the biological safety in the vaccine production process is greatly reduced. It is an ideal candidate vaccine for upgrading the current type D Clostridium perfringens toxin vaccine in my country; and when preparing a combined vaccine with other antigens, the combined vaccine can be prepared without increasing the dosage of the combined vaccine.

The invention discloses a humanized single-chain antibody 8B of Clostridium perfringens α toxin, which is screened from the phage antibody library Source bioscience, and is a fully human anti-Clostridium perfringens α toxin antibody, which can be realized Neutralizing the therapeutic effect of toxins, while overcoming various side effects of heterologous antibodies, eliminating the cumbersome steps and high cost of humanized transformation of heterologous antibodies, due to the small molecular weight of single-chain antibodies and strong penetrating ability in vivo, It quickly reaches damaged tissues and cells to play an anti-toxin effect, thus achieving the dual purpose of economy and high efficiency. The α-toxin has certain homology with other types of toxins of Clostridium perfringens, and the antibody drug may have inhibitory and therapeutic effects on other types of toxins, and can also be used as a detection reagent to detect α-toxins of Clostridium perfringens.