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Method of preparing A, B, E and F type tetravalent botulinum antitoxins

A technology of botulinum toxin type A and botulinum toxin, which is applied in the directions of antitoxins, chemical instruments and methods, antibodies, etc., and can solve problems such as low specific activity, low proportion of neutralizing antibodies in active ingredients, and influence of antitoxin neutralizing activity.

Active Publication Date: 2018-11-16
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, there are too many non-neutralizing epitopes in the toxoid itself, so although the titer of antibodies produced by immunity can be high, the proportion of neutralizing antibodies in the active ingredient is low, that is, the specific activity is relatively low, and the neutralization of the anti-toxin is relatively low. Activity is affected, and the most important indicator to measure the efficacy of antitoxin is the activity of neutralizing antibody (specific activity)

Method used

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  • Method of preparing A, B, E and F type tetravalent botulinum antitoxins
  • Method of preparing A, B, E and F type tetravalent botulinum antitoxins
  • Method of preparing A, B, E and F type tetravalent botulinum antitoxins

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1, Pilot-scale preparation of various types of botulinum toxin recombinant proteins and quality evaluation standards

[0046] Botulinum toxin type A recombinant Hc protein: the coding gene is shown in SEQ ID No.5 (after codon optimization), encoding the protein shown in SEQ ID No.1 (named AHc).

[0047] Recombinant Hc protein of botulinum toxin type B: the coding gene is shown in SEQ ID No.6 (after codon optimization), encoding the protein shown in SEQ ID No.2 (named BHc).

[0048] Type E botulinum toxin recombinant Hc protein: the coding gene is shown in SEQ ID No.7 (after codon optimization), encoding the protein shown in SEQ ID No.3 (named EHc).

[0049] Type F botulinum toxin recombinant Hc protein: the coding gene is shown in SEQ ID No.8 (after codon optimization), encoding the protein shown in SEQ ID No.4 (named FHc).

[0050] 1. Pilot scale preparation of recombinant Hc protein of type A, E and F botulinum toxin

[0051] A, E, and F-type recombinant Hc ...

Embodiment 2

[0060] Example 2. Establishing an immunization scheme using recombinant Hc antigen as an immunogen to prepare high-titer horse anti-botulinum toxin plasma

[0061] Various types of recombinant Hc antigens prepared by the pilot process in Example 1 were used as immunogens for immunizing horses. The specific immunization scheme is designed as follows:

[0062] Source of horses: from non-epidemic areas (no melioidosis, no equine infectious anemia, no brucellosis, no equine paratyphoid abortion), 4-6 years old, good body condition, healthy male, body weight 300-350Kg. According to the requirements of the Pharmacopoeia, the four important infectious diseases of equine poverty, melioidosis, brucellosis and paratyphoid were detected. At the same time, the horse serum was tested for anti-botulinum toxin antibody type A, and the results were all negative, which was in line with the production of antiserum. requirements. And the horses are managed and raised in pens by special personn...

Embodiment 3

[0066] Example 3, Collection, Preparation and Detection of Anti-Botulinum Toxin Type A, B, E and F High Titer Immune Plasma

[0067] The detection method of neutralizing antibody in horse plasma refers to the pharmacopoeia of the People's Republic of China 2015 edition 3510 botulinum antitoxin potency determination method (mouse determination method). The experimental method is briefly introduced as follows:

[0068] ① Dilute the toxin with the sample to be tested. Dilute botulinum toxin types A and B to 1L + / ml (see Pharmacopoeia for details, refers to the toxic dose of toxin, 1L + Equivalent to 10000 LD 50 ), botulinum toxin type E diluted to 0.1L + / ml, botulinum toxin type F diluted to 0.25L + / ml. Then dilute the horse plasma to the equivalent multiple of the corresponding botulinum toxin according to the expected titer, for example, if the horse plasma is expected to be 1000IU / ml, the type A and B botulinum toxins are diluted to 1IU / ml, and the type E botulinum to...

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Abstract

The invention discloses a method of preparing A, B, E and F type tetravalent botulinum antitoxins. The method comprises the following steps: immunizing animals with four immunogens (A, B, E and F typebotulinum toxin recombinant Hc proteins), to separately prepare four monovalent botulinum antitoxins; and mixing the four monovalent botulinum antitoxins to prepare the A, B, E and F type tetravalentbotulinum antitoxins. A novel technical scheme and a pilot process of botulinum toxin recombinant Hc immunogen immune horses to prepare botulinum toxin therapeutic neutralizing antibodies are established. High purity and high valence botulinum antitoxins are prepared through a refining process. The key problem that horse botulinum antitoxin therapeutic antibodies are not prepared by taking toxoids as the immunogens is solved, and the neutralizing valence of the ntitoxins and the quality of the products are improved. The tetravalent botulinum antitoxins prepared by the method can be used for treating poisoning of various creotoxins comprehensively.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for preparing A, B, E, and F type tetravalent botulinum antitoxins. Background technique [0002] Botulinum toxin is an exotoxin produced by Clostridium botulinum. It is currently the most toxic protein known. It is divided into seven serotypes (A-G), of which the naturally occurring A, B, E and F toxins can cause human poisoning. Types C and D often cause poisoning in animals and poultry, and there are fewer reports of human poisoning caused by type G. Botulinum toxin poisoning has a small incidence rate in the crowd, but its toxicity is high, the fatality rate is high, and it is extremely harmful to public health safety, so people must pay great attention to it. From the results of primate experiments, it is estimated that the semi-lethal dose of human botulinum toxin poisoning with a body weight of 70 kg is 0.09-0.15 μg for intravenous or intramuscular injection, 0.7-...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/06A61K39/40A61P39/02
CPCA61K2039/507A61P39/02C07K16/06C07K16/1282C07K2317/35
Inventor 余云舟师丹阳杨志新陆建昇王荣周晓巍
Owner ACADEMY OF MILITARY MEDICAL SCI
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