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Protein sequence 12G12 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 12G12 and application thereof
  • Protein sequence 12G12 and application thereof
  • Protein sequence 12G12 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of PET-24a / 12G12 expression strain

[0035] The 12G12 protein sequence (SEQ ID NO:1) is converted into a DNA sequence and optimized (choose the commonly used codons in the Ecoli system, and balance the distribution of GC bases to make the distribution uniform, reduce the probability of secondary structure formation, etc.) After the sequence (SEQ ID NO: 2), the NdeI (CATATG) and XhoI (CTCGAG) double enzyme cutting sites were added at the front and rear ends of SEQ ID NO: 2, and then the SEQ ID NO: 3 was artificially synthesized (for Suzhou Jinweizhi Biological Science and Technology Co., Ltd.), the synthesized sequence SEQ IDNO: 3 was double-enzyme digested (NdeI and XhoI) and then ligated to the same double-enzyme-cut pET-24a vector, and then transferred into DH5α bacteria 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 screened by Kanamyci...

Embodiment 2

[0037] Example 2: 12G12 expression strain expansion culture and protein purification

[0038] The expression strain obtained in Example 1 was recovered through the following steps, seed expansion and IPTG induced fermentation and purification finally obtained purity> More than 90% of the target protein, the SDS-PAGE results of the purified protein are shown in detail image 3 . Lane 1 is the purified 12G12 protein, and lane 2 is the molecular standard protein. It can be seen from the SDS-PAGE electrophoresis that the cell supernatant expressed in the 12G12 bacterial cell has a purity of more than 90% after purification by a nickel column.

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

[0040] (2) Pick a single colony and inoculate it ...

Embodiment 3

[0048] Example 3: In vitro activity determination of 12G12

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

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

[0051] 1) Cell sample preparation: collect 1×10 in a 2ml round-bottomed EP tube 6 Cells; centrifuge at 800rpm for 5min; discard the supernatant and add 1ml sterile PBS to wash the cell pellet; centrifuge at 800rpm for 5min; discard the supernatant and add 100μl of 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: Add Anti-AXL antibody to the cell suspension and incubate at 4°C for 60 min.

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

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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 12G12 and its uses. Background technique [0002] The AXL receptor is a member of the tyrosine protein kinase family and is considered a potential target for tumor therapy. In many cancer patients, the cell signaling pathways activated by AXL are usually related to the promotion of tumor metastasis, tumor resistance and disease progression. And it is 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 the outcome. 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 looking for AXL inhibitors. [0003] At present, the publicly reported or under research AXL inhibitors are roughly divided into two categories: 1. AXL small molecule inhibitors, the m...

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 张文宇白羊葛平辉张聪慧王明灶
Owner SINOBIOWAY BIOMEDICINE
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