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Recombinant superantigen SEB mutant, preparation method and applications thereof

A mutant and superantigen technology, applied in the field of medicine and biology, can solve the problems of reduced anti-tumor activity, T-cell incompetence, toxic shock, etc., and achieve the effect of enhanced anti-tumor effect, simple and easy operation, and enhanced activity.

Active Publication Date: 2014-08-06
军事科学院军事医学研究院微生物流行病研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Thanks to SE s It is a bacterial exotoxin, which can cause food poisoning at a certain concentration, and may also cause symptoms such as fever, vomiting, diarrhea, and toxic shock
In recent years, studies have shown that superantigens can produce immunosuppressive effects in both in vivo and in vitro tests, and high doses and repeated administration can lead to T cell incompetence, resulting in reduced anti-tumor activity, which limits its clinical application to some extent. Application in tumor immunotherapy
However, there is no report on the multi-point mutation of the superantigen SEB to reduce toxic side effects and enhance anti-tumor activity.

Method used

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  • Recombinant superantigen SEB mutant, preparation method and applications thereof
  • Recombinant superantigen SEB mutant, preparation method and applications thereof
  • Recombinant superantigen SEB mutant, preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preparation of embodiment one recombinant superantigen SEB mutant

[0026] 1. Materials:

[0027] 1.1 Main Instruments

[0028] PCR amplification instrument (Whatman Biometra, Germany), electric thermostatic water bath (Beijing Changfeng Instrument Co., Ltd.), AKTA FPLC protein purification instrument (GE, USA), polyacrylamide gel electrophoresis apparatus (BIORAD, USA)

[0029] 1.2 Strains and main reagents

[0030] Natural Staphylococcus aureus enterotoxin SEB strain ATCC (14458), purchased from ATCC (American Standard Biological Collection); pET-32(a+) expression vector, purchased from Novagen; Escherichia coli DH5α, BL21(DE3) It is a product of Invitrogen Company; gene cloning vector pGEM-T was purchased from Promega Company; NdeI and BamHI were purchased from TaKaRa Biotechnology Company; T4 DNA ligase was purchased from New England BioLabs Company; IPTG was purchased from AMRESCO Company; Guangzhou Dongsheng Company; Genomic DNA Extraction Kit, Saibaisheng ...

Embodiment 2

[0060] Western-blot identification of embodiment two purified proteins

[0061] 1. Materials:

[0062] 1.1 Main Instruments

[0063] ST-1 semi-dry transfer electrophoresis tank, Dalian Jingmai Biotechnology Co., Ltd.

[0064] 1.2 Main reagents

[0065] Mouse anti-wt-SEB monoclonal antibody, goat anti-mouse IgG / HRP were purchased from SANTA CRUZ Company; bovine serum albumin (BSA) was purchased from Beijing Biocom Biotechnology Company; SDS and ECL developer were purchased from Sigma, USA; super West Pico Trial Kit was purchased from Thermo Company. The remaining is the same as previous embodiment.

[0066] 2. Method results:

[0067] The prepared protein was electrophoresed by SDS-PAGE and transferred to the membrane by semi-dry method. at 1mA / cm2 The power, constant current transfer 1h. After the transfer, the nitrocellulose membrane was removed and blocked overnight at 4°C with 5% skimmed milk powder. After the blocking, the blocking solution was discarded, and the...

Embodiment 3

[0068] Example 3 Cytotoxic activity test (MTS detection)

[0069] 1. Materials:

[0070] 1.1 Main Instruments

[0071] Bx60-32FB2-E01 fluorescence microscope, Olympus, Japan; ELISA, USA

[0072] National SPECTRA MAX PLUS

[0073] 1.2 Main reagents and consumables

[0074] MTS detection kit was purchased from Promega; cell culture flasks, culture plates and other consumables and RPMI1640 medium were purchased from GIBCO; Ficoll-PaqueTM PLUS lymphocyte separation medium was purchased from GE

[0075] 1.3 Cell lines

[0076] Human tumor cells (human liver cancer cell line SMMC-7721, cervical cancer cell line Hela, gastric cancer cell line BGC-823), normal human brain microvascular endothelial cells HBMEC were purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The remaining is the same as previous embodiment.

[0077] 2. Method results:

[0078] Take various human cells in good condition, the cell viability measur...

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Abstract

The present invention discloses a recombinant superantigen staphylococcal enterotoxin B (SEB) mutant, and a preparation method and applications thereof. According to the present invention, a SEB genome derived from natural staphylococcus aureus is extracted, and after the SEB genome is obtained, site-specific mutagenesis at toxicity-related amino acid sites is performed to obtain the SEB mutant. The SEB mutant has characteristics of both enhanced activity and weakened toxicity, and can be used as anti-tumor immune therapy drugs or immune system modulating drugs. The recombinant superantigen SEB mutant has good market prospects.

Description

Technical field: [0001] The invention belongs to the technical field of medicine and biology, and relates to a staphylococcus aureus enterotoxin, in particular to a type B staphylococcus aureus enterotoxin (SEB), and also relates to its preparation method and application. Background technique: [0002] At present, more than 30,000 natural products have been isolated from the metabolites of microorganisms, a large part of which are biologically active, and they are expected to be developed into drugs. There are more than 100 microorganism-related drugs that have been released. Superantigen (SAg) is a high-efficiency immunomodulatory protein, which does not need to be processed by antigen-presenting cells, but directly binds to the outside of the MHC-II antigen-binding groove of antigen-presenting cells and the T cell receptor Vβ fragment. APC forms MHC-SAg-TCR ternary complex to activate T cells, and its ability to induce strong proliferation and differentiation of T lymphoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70C12N1/21A61K38/16A61P35/00A61P37/02C12R1/445C12R1/19
CPCC07K14/31C12N15/70
Inventor 姜永强江华郑玉玲谷丽维郑欣王君王燕子
Owner 军事科学院军事医学研究院微生物流行病研究所
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