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Protein sequence 11B09 and application thereof

A protein sequence and application technology, applied in the field of gene sequence, can solve the problems of complex production process, poor specificity, and many negative effects, and achieve the effect of inhibiting tumor cell activity, overcoming poor specificity, and overcoming complex production process

Inactive Publication Date: 2019-01-15
SINOBIOWAY BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two types of AXL inhibitors have their own disadvantages: 1. AXL small molecule inhibitors have poor specificity, so when inhibiting the target of AXL, they will also inhibit other similar targets, and have more negative effects
2. AXL antibodies and antibody analogs are glycoproteins, so they are mainly expressed in mammalian cells during the production process, the production process is complex, and the quality control and production costs are high

Method used

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  • Protein sequence 11B09 and application thereof
  • Protein sequence 11B09 and application thereof
  • Protein sequence 11B09 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of PET-24a / 11B09 expression strain

[0035] The protein sequence (SEQ ID NO:1) of 11B09 was converted into a DNA sequence and optimized (select the codons commonly used in the Ecoli system, and at the same time balance the distribution of GC bases to make them evenly distributed and reduce the probability of secondary structure formation, etc.) The following sequence (SEQ ID NO: 2), artificially synthesized SEQ ID NO: 3 after adding NdeI (CATATG) and XhoI (CTCGAG) double restriction sites at the front and rear ends of SEQ ID NO: 2 respectively (given to Suzhou Jinweizhi Biological Technology Co., Ltd.), the synthesized sequence SEQ IDNO: 3 double enzyme digestion (NdeI and XhoI) is connected to the pET-24a vector of the same double enzyme digestion, and then transferred to DH5α cells to amplify and extract the plasmid to obtain the expression vector .

[0036] Then the expression vector was transformed into BL21, and the positive strain was s...

Embodiment 2

[0037] Example 2: Expanded culture and protein purification of 11B09 expression strain

[0038] The expression strain obtained in Example 1 was recovered through the following steps, seed expansion, IPTG-induced fermentation and purification to finally obtain the target protein with a purity of >90%. The purified protein SDS-PAGE results are detailed in image 3 . Among them, lane 1 is purified 11BO9 protein, and lane 2 is molecular standard protein. From the SDS-PAGE electrophoresis figure, it can be seen that the purity of the target protein in the cell supernatant expressed in the 11BO9 cell reaches more than 90% after being purified by a nickel column.

[0039](1) Dilute the target glycerol bacterium frozen at -80°C with LB liquid medium 100,000 times, spread on the agar plate with 50 μg / ml Kan (or scrape the glycerol bacterium with a sterilized pipette tip, scratch line on the agar plate), and cultured upside down in a 37°C incubator overnight (about 16h);

[0040] (2)...

Embodiment 3

[0048] Example 3: 11B09 in vitro activity assay

[0049] 1. FACS screening and verification of AXL high expression cell lines

[0050] Through relevant literature research, the applicant selected candidate cell lines with high expression of AXL: A549-luc, MDA-MB-231-luc, SKOV3, NCI-H1975-luc, and verified them by FACS. The specific test protocol is as follows:

[0051] 1) Cell sample preparation: collect 1×10 cells in a 2ml round bottom EP tube 6 centrifuge at 800rpm for 5min; discard the supernatant, add 1ml sterile PBS to wash the cell pellet; centrifuge at 800rpm for 5min; discard the supernatant, add 100μl sterile PBS to resuspend the cells; transfer the cell suspension to a 96-well round bottom plate.

[0052] 2) Primary antibody (Anti-AXL antibody) incubation: Anti-AXL antibody was added to the cell suspension and incubated at 4°C for 60 min.

[0053] 3) Wash Anti-AXL antibody: centrifuge the 96-well round bottom plate at 2000rpm×3min, discard the supernatant; add 200μ...

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Abstract

The invention discloses a protein sequence. The sequence is as shown in SEQ ID No. 1. A corresponding nucleic acid sequence is as shown in SEQ ID No. 2. The protein sequence is applicable as an AXL inhibitor and can inhibit the phosphorylation of AXL in A549 and MDA-MB-231 cells. The invention provides the application of the protein sequence to inhibition of the phosphorylation of AXL in cells andfurther provides the application of the protein sequence to inhibition of the activity of tumor cells.

Description

technical field [0001] The present invention relates to the field of gene sequences, in particular to the protein sequence 11B09 and its use. Background technique [0002] AXL receptor is one of the members of the tyrosine protein kinase family and is considered as a potential target for tumor therapy. In many cancer patients, the cell signaling pathways activated by AXL are usually associated with the promotion of tumor metastasis, tumor drug resistance and disease progression. It is also found to be highly expressed in a variety of solid tumors and leukemia cancer cells, and its expression level is positively correlated with the disease process and prognosis. Therefore, AXL can be used as an important target for tumor therapy, and it is of great significance to find new therapeutic approaches for tumor therapy by finding AXL inhibitors. [0003] At present, AXL inhibitors publicly reported or under research can be roughly divided into two categories: 1. AXL small molecul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/70A61K38/16A61P35/00
CPCA61K38/00A61P35/00C07K14/001C12N15/70
Inventor 张文宇葛平辉朱长发邹有土白羊
Owner SINOBIOWAY BIOMEDICINE
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