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Anti-bladder cancer target superantigen and preparation method thereof

A technology of bladder cancer and superantigen, which is applied in the field of anti-bladder cancer targeted superantigen and its preparation, can solve the problems of poor therapeutic effect of bladder cancer, high toxicity and side effects, and weak specificity, so as to avoid the disorder of the body's immune regulation and Toxin poisoning syndrome, simplifying the preparation process, and overcoming the general narrowing effect of anti-tumor

Inactive Publication Date: 2012-05-23
XUZHOU CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the disadvantages of current bladder cancer perfusion therapy, such as poor therapeutic effect, large toxic and side effects, narrow anti-tumor effect, and weak specificity, the present invention provides a simple and efficient anti-bladder cancer targeting superantigen applied to bladder perfusion and a preparation method thereof

Method used

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  • Anti-bladder cancer target superantigen and preparation method thereof
  • Anti-bladder cancer target superantigen and preparation method thereof
  • Anti-bladder cancer target superantigen and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1BD

[0044] Example 1 Preparation of BDI-1Fab-SEA anti-bladder cancer targeting superantigen

[0045] Preparation steps:

[0046] 1. Preparation and purification of monoclonal antibody BDI-1 Fab fragment

[0047] 10ml of BDI-1 and 50μl of papain, add β-mercaptoethanol to a final concentration of 20μM, react at 37°C for 3h to obtain Fab, dialyze with PBS overnight; purify the dialyzed sample on a protein G column, wash the column with PBS and collect the effluent peak ; Concentrate by centrifuging a 15ml ultrafiltration centrifuge tube (pore size 80000D) at 2000G for 1h; add 500μl double distilled water and 50μl Bacine (1M, pH9.0) to obtain 1ml of 0.1M Bacine system with pH8.5 for use.

[0048] In this step, BDI is digested with papain and protein G chromatography to obtain a purified Fab fragment product.

[0049] 2. Preparation of BDI-1Fab-SEA anti-bladder cancer targeting superantigen with chemical linker SPDP:

[0050] Dissolve 0.24mg of SEA in 400μl of PBS, take 1ml of the a...

Embodiment 2

[0055] Identification of each collection peak molecular weight and purity of the protein liquid chromatography system of embodiment 2

[0056] Using 8% concentration gel, SDS-PAGE discontinuous system, Coomassie brilliant blue staining, using SEA, Fab standard protein control, to identify the relative molecular mass and purity of each collected peak protein see figure 2 .

[0057] Such as figure 2 In, A is the electropherogram of the sample before purification: Lane is Marker, the maximum molecular weight is 96kd; Lane2 is the sample before purification of anti-bladder cancer targeted superantigen; Lane3 is the SEA standard product, the molecular weight is about 28kd; Lane4 is the Fab standard product, the molecular weight is about 50kd . The results showed that the pre-purification samples also included proteins with larger molecular weights in addition to SEA and Fab, again suggesting that the preparation was successful. .B. is the electropherogram of the purified first...

Embodiment 3

[0059] Example 3 Anti-bladder cancer targeting superantigen targeting activity identification

[0060] Bladder cancer cell line BIU-87 slides were used as antigen sheets, anti-bladder cancer targeted superantigen samples diluted in urine were used as the primary antibody, anti-SEA rabbit McAb was used as the secondary antibody, non-biotinylated rabbit secondary antibody, DAB was used as the third antibody Color development; Lovo cell slides were used instead of antigen sheets, and SEA and PBS were used instead of primary antibodies as controls.

[0061] The prepared anti-bladder cancer targeting superantigen is still anchored on the surface of bladder tumor cells after dilution in urine, such as image 3 A, using anti-bladder cancer targeting superantigen as the primary antibody, the cell membrane of BIU-87 cells can be stained with brownish yellow positive staining (×400); image 3 B, C, PBS instead of the primary antibody control group showed no positive staining on the cel...

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Abstract

The invention relates to an anti-bladder carcinoma targeted superantigen, which comprises a targeted part and a superantigen part, wherein the targeted part is a BDI-1 fragment, capable of being specially combined with bladder carcinoma cells, of an anti-bladder carcinoma monoclonal antibody, and the superantigen part is Staphylococcus aureus enterotoxin A (SEA); and the targeted part and the superantigen part are connected into the following general formula through a chemical connecting agent 3-(2-pyridine dimercapto)metacetonic acid N-hydroxysuccinimide eater: (BDI-1Fab-NHOCCH2CH2SSCH2CH2CONH)n-SEA, wherein n is less than or equal to 7. The targeted superantigen not only maintains the targeting property of BDI-1Fab on the bladder tumor cells but also maintains the strong functions of the SEA in activating T lymphocytes and killing the bladder tumor cells, has obvious treatment effect, and is safe and harmfulless when being applied to bladder irrigation treatment. The invention adopts the chemical coupling method to prepare Fab-SEA, simplifies the preparation technology, and has convenient industrial production.

Description

technical field [0001] The invention relates to an anti-bladder cancer targeting superantigen applied to bladder perfusion and a preparation method thereof. Background technique [0002] The incidence of bladder cancer ranks first in the malignant tumors of the genitourinary system in my country, and it is increasing year by year. More than 90% of bladder cancers are transitional cell carcinomas (TCC), and about 75-85% of them are superficial bladder cancers (superficial bladder cancers, SBCs) confined to the mucosal layer and lamina propria. For SBC patients, TURBT and partial cystectomy can be used; however, 10%-67% of patients relapse within 12 months after operation, and 24%-84% of patients relapse or even progress within 5 years. Therefore, postoperative intravesical chemotherapy or immunotherapy is the standard treatment for SBC. [0003] However, intravesical infusion chemotherapy is different from systemic chemotherapy. The curative effect of drugs is not directly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/14C12P21/08
Inventor 韩从辉贡震郝林
Owner XUZHOU CENT HOSPITAL
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