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Preparation method and application of E type botulinum toxin recombinant HN-L antigen

A botulinum toxin and antigen technology, applied in the direction of recombinant DNA technology, bacterial antigen components, chemical instruments and methods, etc., can solve the problems that cannot be popularized and applied, and achieve the effect of strong immune activity

Active Publication Date: 2020-03-31
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Botulinum toxin is currently the most toxic protein known, and the development of vaccines and neutralizing antibodies has attracted much attention. However, the current research or limited use of toxoid vaccines have many shortcomings and cannot be widely used

Method used

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  • Preparation method and application of E type botulinum toxin recombinant HN-L antigen
  • Preparation method and application of E type botulinum toxin recombinant HN-L antigen
  • Preparation method and application of E type botulinum toxin recombinant HN-L antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Expression, purification and identification of recombinant antigens with functional epitopes of type E botulinum toxin in Escherichia coli

[0052] 1. Gene design and synthesis of each functional epitope antigen of botulinum toxin type E

[0053] According to the codon degeneracy, the genes encoding the functional epitopes of E-type botulinum toxin were artificially optimized and synthesized, and first cloned directly into the pMD18-T vector (TaKaRa) and named as pMD18-L, pMD18-HN, pMD18-HN- L, pMD18-Hc, pMD18-Hc-C, and pMD18-Hc-N encode L, HN, HN-L, Hc, Hc-C, and Hc-N, respectively (see Table 1 for detailed antigen and sequence information). When cloning these artificially synthesized genes, in order to facilitate the following operations, the 5' end of each functional epitope antigen gene was introduced into EcoR I and the 3' end introduced the Xho I restriction recognition site.

[0054] The above gene design adopts the codons commonly used in Escherichia...

Embodiment 2

[0067] Example 2 Detection of Antibody Levels and Protective Effects on Type E Botulinum Toxin After Immunizing Mice with Various Functional Epitope Antigen Proteins in the Carboxy-terminal Structural Region of the Heavy Chain of Botulinum Toxin Type E

[0068] The recombinant antigens prepared in Example 1 above were immunized into mice respectively, and their immunogenicity and protective properties were tested. The specific method is as follows: Balb / c mice (6-8 weeks, female, SPF grade, purchased from the Experimental Animal Center of the Military Medical Research Institute) were randomly divided into groups, with 8 mice in each group, and the immunization dose was 1 μg or 10 μg per mouse, respectively. Antigen protein, each antigen protein in the combined group was half and half, while the negative control group was immunized with PBS not containing recombinant protein, and aluminum adjuvant (Alhydrogel) with a final concentration of 1mg / ml TM 2.0%, Bom Tai (Brenntag Bios...

Embodiment 3

[0078] Example 3: Detection of Antibody Levels and Protective Effects on Type E Botulinum Toxin After Immunizing Mice with Various Functional Epitope Antigen Proteins in the Heavy Chain N-Terminal-Light Chain Structural Region of Type E Botulinum Toxin

[0079] The immunization scheme for the epitope antigen protein of the N-terminal of heavy chain of type E botulinum toxin-light chain structural region is the same as that of Example 2. Table 3 and image 3 The results shown show that HN, EL and HN-L antigens were immunized with 1 and 10 μg doses for 2 and 3 times, and each recombinant antigen produced an antibody response only against itself, without other cross-antibody reactions. Different doses and times of immunization in each immunization group produced protective responses, and also showed a dose-positive correlation. However, it is worth noting that the protective effect of the HN-L antigen was higher than that of the HN and L groups, especially at low doses. At the ...

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Abstract

The invention discloses a preparation method and application of an E type botulinum toxin recombinant HN-L antigen. The invention provides application of an E type botulinum toxin recombinant HN-L antigen (a light chain and heavy chain amino acid fusion region of E type botulinum toxin) in any one of the following aspects: application in preparation of an E type botulinum toxin subunit vaccine andapplication of the E type botulinum toxin recombinant HN-L antigen as immunogen in preparation of E type botulinum antitoxin. The experiment proves that the efficacy of the HN-L antigen as the immunogen is superior to the efficacy of other functional domain antigens, including the Hc antigen, as immunogens. The characteristic of the HN-L antigen enables a problem that the E type botulinum toxin recombinant Hc antigen is not strong enough in immune protection. In addition, the immunogen with stronger immune efficacy is required in clinical tests of human bodies so as to generate strong immunocompetence. Therefore, the E type botulinum toxin recombinant HN-L antigen disclosed by the invention is expected to be used as a candidate subunit vaccine to be applied to prevention of E type botulinum toxin.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a preparation method and application of E-type botulinum toxin recombinant HN-L antigen. Background technique [0002] Botulism is caused by neurotoxins (BoNTs) secreted by the bacterium botulinum. Botulinum toxin is the most toxic substance among known biological and chemical poisons to human beings. 50 Only 0.1-1ng / kg, according to different serological properties, botulinum toxin is divided into 7 types A ~ G, of which A, B and E cause more human poisoning, and F type is less. Botulinum toxin has been prepared as a biological warfare agent by many countries, and it has also attracted strong interest from terrorist organizations. Therefore, botulinum toxin is listed as one of the six most important biological warfare agents by the US CDC and other institutions, and belongs to the most harmful Class A biological agent. Botulinum toxin poisoning has a small incidence rate in th...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/12C12N15/62C12N15/70A61K39/08A61P31/04A61P39/02
CPCA61K39/08A61P31/04A61P39/02C07K14/33C07K16/1282C07K2319/00C12N15/70
Inventor 余云舟杨志新陆健昇王荣
Owner ACADEMY OF MILITARY MEDICAL SCI
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