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Preparation method and application of e-type botulinum toxin recombinant l-hn antigen

A botulinum toxin and antigen technology, applied in recombinant DNA technology, bacterial antigen components, chemical instruments and methods, etc., can solve problems such as inability to popularize and apply

Active Publication Date: 2021-08-31
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 l-hn antigen
  • Preparation method and application of e-type botulinum toxin recombinant l-hn antigen
  • Preparation method and application of e-type botulinum toxin recombinant l-hn 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-L- HN, pMD18-Hc, pMD18-Hc-C, and pMD18-Hc-N encode L, HN, L-HN, 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 showed that HN, EL and L-HN antigens were immunized with 1 and 10 μg doses for 2 and 3 times, and each recombinant antigen produced antibody responses only against itself, without other cross-antibody responses. 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 L-HN antigen is higher than that of HN and L groups, especially the difference is more obvious ...

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Abstract

The invention discloses a preparation method and application of E-type botulinum toxin recombinant L-HN antigen. The present invention provides the application of E-type botulinum toxin recombinant L-HN antigen (light chain and heavy chain amino acid fusion region of E-type botulinum toxin) in any of the following applications: the application in the preparation of E-type botulinum toxin subunit vaccine ; As an immunogen in the preparation of E-type botulinum antitoxin application. The present invention demonstrates that the effectiveness of the L-HN antigen as an immunogen is superior to that of other functional domain antigens including the Hc antigen. This feature just makes up for the problem that the E-type botulinum toxin recombinant Hc antigen is not effective enough in terms of immune protection. In addition, human clinical trials may require more potent immunogens in order to be able to generate strong immune activity. Therefore, the E-type botulinum toxin recombinant L-HN antigen of the present invention is very promising as a candidate subunit vaccine for the 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 L-HN 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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Patent Type & Authority Patents(China)
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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