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Polypeptide in specific binding with breast cancer brain metastases cells

A technology for brain metastases and breast cancer, applied in the field of preparation of the polypeptide, can solve the problems of specific binding of brain metastases of breast cancer cells that are not mentioned, and achieve the effect of enhancing anti-tumor effect, high specificity and small molecular weight

Active Publication Date: 2013-06-12
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although both patent documents are polypeptides related to the treatment of breast cancer, neither of them mentioned that they can specifically bind to breast cancer brain metastasis cells

Method used

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  • Polypeptide in specific binding with breast cancer brain metastases cells
  • Polypeptide in specific binding with breast cancer brain metastases cells
  • Polypeptide in specific binding with breast cancer brain metastases cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0059] Screening and preparation of embodiment 1 BrBP1 polypeptide

[0060] The present invention screens and prepares BrBP1 polypeptide through the following methods and steps.

[0061] 1. Preliminary screening of phage clones that bind to breast cancer brain metastases cell lines

[0062] Phage display technology was used to conduct multiple rounds of whole-cell subtractive screening, and the phage clones that combined with the breast cancer brain metastases cell line MDA-231BR were preliminarily screened out. The specific steps are as follows:

[0063] In the first round of screening, only the breast cancer brain metastases cell line MDA-231BR was used for positive screening. Breast cancer brain metastases cell line MDA-231BR cells were digested with trypsin, and the cell density was adjusted to 2.5×10 5 / ml, inoculated in cell culture dish (60×15mm 3 ), when the cells grow to 80%-90% confluence, screening is carried out.

[0064] The specific steps of screening are as ...

Embodiment 2B

[0088] Bioinformatics analysis of embodiment 2 BrBP1 polypeptide

[0089] In this example, the ProtParam tool provided by the ExPASy website (http: / / web.expasy.org / protparam / ) was used to analyze the physical and chemical properties of the above-mentioned BrBP1 polypeptide. As shown in Table 2, the molecular weight of the polypeptide is 1487.6, the theoretical isoelectric point is 4.00, it is composed of 199 atoms, the extinction coefficient is 5.700, and the half-life in mammalian reticulocytes is about 30h. is 32.50, and the overall average hydrophobicity is -0.858.

[0090] Table 2 Analysis of physical and chemical characteristics of BrBP1

[0091]

[0092]

Embodiment 3B

[0093] Example 3 The specificity of BrBP1 polypeptide binding to breast cancer brain metastases cell lines

[0094] 1. Identify the combination of phage clones and various cell lines by ELISA

[0095] In this experiment, the No. 89 phage clone (named Phage89) carrying the BrBP1 fragment was selected, and the binding ability of Phage89 to different cell lines was identified by ELISA. Breast cancer brain metastasis cell MDA-231BR, breast cancer parental cell line MDA-231P, breast cancer cell line MCF-7, ovarian cancer SK-OV-3, pancreatic cancer PANC-1, gastric cancer BGC-823, colon cancer HT- 29. Prostate cancer PC-3, liver cancer SMMC7721 cells, human embryonic liver cell line HL-7702, human gastric mucosal immortalized cell line GES-1, and human embryonic kidney cell line 293T were digested with 1×10 trypsin 4 Cells were inoculated in a 96-well plate at a density of 24 hours, treated without serum for 1 hour, washed with PBS three times, and fixed with 4% paraformaldehyde / PBS...

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Abstract

The invention discloses BrBP1 polypeptide in specific binding with breast cancer brain metastases cells, and further relates to a preparation method of the polypeptide. The method includes: by utilization of phage display technology, carrying out a plurality of rounds of whole-cell depletion screening, and screening out phage clones bound with a breast cancer brain metastases cell line; randomly picking out a plurality of the phage clones, and selecting strongly positive phage clones with a cell enzyme-linked immuno sorbent assay (ELISA) method; and after increasing the strongly positive phage clones, carrying out sequencing, and synthesizing the tested BrBP1 polypeptide in a manual mode. The polypeptide can be applied to preparation of medicine used for curing breast cancer brain metastases and kits used for early-stage diagnosis of the breast cancer brain metastases. The polypeptide can be synthesized with a manual method, is small in molecular weight, high in activity, strong in penetrating power, high in affinity and specificity and low in toxicity, has good tumor-targeting performance inside and outside a body, can also be internalized to enter cells, and is suitable for being used as vectors for targeted therapy.

Description

technical field [0001] The present invention relates to a polypeptide for treating tumors, especially a polypeptide that specifically binds to breast cancer brain metastases cells. The present invention also relates to a method for preparing the polypeptide, and the polypeptide is used in the preparation of drugs for treating breast cancer brain metastases, The preparation is used in the early diagnosis kit of breast cancer brain metastases. Background technique [0002] Breast cancer is a malignant tumor that seriously endangers women's health. The morbidity and mortality of breast cancer worldwide rank first among female malignancies, accounting for 23% of all female cancer cases and 14% of cancer deaths. The typical manifestation of advanced breast cancer is distant spread and metastasis, mainly to bone and brain. Among them, the incidence of brain metastasis of breast cancer accounts for 10%-16% of metastatic cases. In recent years, with the prolongation of patient su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/00A61K38/10A61P35/04G01N33/68
Inventor 张建琼付波张莹
Owner SOUTHEAST UNIV
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