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Targeted function superantigen and its preparation method and use

A superantigen, functional technology, applied in the field of superantigens

Inactive Publication Date: 2006-12-27
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is also no report on the fusion of B3 disulfide bond-stabilized single-chain antibody sc-dsFv with other proteins (toxins, superantigens)

Method used

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  • Targeted function superantigen and its preparation method and use
  • Targeted function superantigen and its preparation method and use
  • Targeted function superantigen and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of B3ds-scFv-SEA fusion protein

[0023] 1. Materials

[0024] The B3ds-scFv-pET and SEA(D227A)-pET plasmids were constructed according to conventional methods. The B3ds-scFv gene fragment was artificially synthesized by Shanghai Boya Biotechnology Co., Ltd., and the SEA(D227A) fragment was caught by Staphylococcus aureus FRI100A (ATCC). And mutagenesis by conventional methods. Human colon cancer cell line HT-29, human liver cancer cell line SMMC-7721, human breast cancer cell line MCF-7, and Escherichia coli TOP10 are all international standard strains, commercially available; expression vector pET22b is commercially available; LA TaqDNA polymerase, All restriction endonucleases were purchased from Takara Company; T 4 DNA ligase, plasmid extraction kit, and Non-RadioactiveCell Proliferation Assay kit were purchased from Promega; DNA purification kit was purchased from Doppler; goat anti-rabbit IgG was purchased from Beijing Xinjingke;...

Embodiment 2

[0052] Example 2 Detection of B3ds-scFv-SEA fusion protein

[0053] 1. Material: same as embodiment one.

[0054] 2. Method

[0055] 2.1 ELISA identification of single-chain antibody activity in B3ds-scFv-SEA

[0056] Tumor cell concentration was adjusted to 5 x 10 with serum-containing 1640 4 / ml spread on 96-well plate, place 100uL per well in a cell culture incubator for 48 hours, take it out, discard the culture medium, wash it once with PBS, and then add 100ul of pre-cooled cell fixative (V methanol: V acetone) to each well =1:1) After fixing for 30 min, add blocking solution (containing 1% H 2 o 2 After blocking with PBS for 10 min, wash three times with PBS; add protein solution (initial concentration: 100 μg) gradiently diluted with PBST containing 3% BSA, react at 37°C for 1 h, add 1:1000 diluted rabbit-derived antibody after washing with PBST SEA primary antibody, combined for 1 hour at 37°C; after washing with PBST, add goat anti-rabbit IgG secon...

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PUM

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Abstract

The invention discloses a super antigen target to liver cancer, and also discloses the method for preparing the same and its usage. Said super antigen comprises target part and super antigen part, said target part is genetic engineering antibody coming from B3 monoclonal antibody and combined with LeY carbohydrate antigen, and the super antigen is staphylococcus enterotoxin A. The preparing method comprises connection of VH and VL section of B3 antibody, construction of recombined carrier containing antibody and super antigen gene section, expression of super original fusion protein and purification. The recombined super antigen is characterized by double function of guide and killing cancer cell, and can be made to effective target medicine for treating tumor.

Description

technical field [0001] The present invention relates to a superantigen with targeting function, in particular to a targeting superantigen containing a disulfide bond-stabilized single-chain antibody, and to a preparation method and application of the superantigen. Background technique [0002] Staphylococcal enterotoxin A (staphylococcalenterotoxinA SEA) is the product of Staphylococcus aureus and is an important bacterial superantigen. Since the CTL (cytotoxic Tlymphocytes) induced by SEA has a powerful killing effect on tumor cells (Kalland T, Dohlsten M, AbrahmsenL, et al. Targeting of superantigens. Cell Biophys. 1993 Jan-Jun; 22(1-3): 147- 164; Hansson J, Ohlsson L, Persson R, et al. Genetically engineered superantigens astolerable antitumor agents. Proc Natl Acad Sci USA. 1997 Mar 18; 94(6): 2489-2494; Dohlsten M., Hedlund G., Akerblom E ., et al. Antibody-targeted superantigens: adifferent class of anti-tumor agents. Proc Natl Acad Sci USA. 1991 October 15; 88(20): 9...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K48/00A61K39/395A61K39/085A61P35/00
Inventor 姜永强郑玉玲王建丽马茹宁保安
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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