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A BMX spliceosome and applications thereof in lung cancer drug resistance

A splicing body, lung cancer cell technology, applied in the biological field, can solve problems such as lack of a good solution strategy and weakening the effect of targeted drug therapy

Active Publication Date: 2016-03-30
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinically, these small molecule inhibitors have a significant effect on lung cancer patients with the above-mentioned EGFR kinase domain mutations, but the emergence of some drug-resistant mutations (such as EGFR kinase domain T790M point mutation, MET or HER2 gene amplification, etc.) has greatly weakened The therapeutic effect of targeted drugs, and currently there is no good solution

Method used

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  • A BMX spliceosome and applications thereof in lung cancer drug resistance
  • A BMX spliceosome and applications thereof in lung cancer drug resistance
  • A BMX spliceosome and applications thereof in lung cancer drug resistance

Examples

Experimental program
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Effect test

preparation example Construction

[0102] The preparation of the BMXΔN chip can adopt conventional manufacturing methods of biochips known in the art. For example, if the solid phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then spotted with a spotting instrument. The BMXΔN chip of the present invention can be obtained by arranging in a predetermined sequence or array on a modified glass slide or a silicon chip, and placing it overnight for fixation.

[0103] Reagent test kit

[0104] The present invention also provides a kit, which includes reagents or chips for measuring BMXΔN;

[0105] Wherein, described reagent is selected from following group:

[0106] (a) a primer or primer pair that specifically amplifies said BMXΔN;

[0107] (b) a probe that specifically hybridizes with the nucleic acid molecule of the BMXΔN;

[0108] Wherein, the chip is a nucleic acid chi...

Embodiment 15

[0155] Example 15' RACE analysis of BMX abnormal splicing sites

[0156] 5' RACE according to SMARTer TMRACE cDNA Amplification Kit (Clontech Laboratories Inc, Mountain View, CA) kit instruction manual operation.

[0157] First, RNA was extracted from lung tumor tissue according to the instructions of the RNeasyMiniKit (Qiagen, Hilden, Germany) kit, and then 1 μg of RNA was taken and reverse-transcribed with the 5' RACECDS primer A and SMARTerIIA oligonucleotide provided by the kit; Universal primer A mix (UPM) 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'(SEQ ID NO.:5) and 5'-CTAATACGACTCACTATAGGGC-3'(SEQ ID NO.:6) coupled BMX gene-specific primer GSP1: 5'-GGTTCAAGTCCTTTTCGTGACTCCTCA-3' (SEQ ID NO.: 7) or GSP2: 5'-CCCGAAGTGGTTCAATGGAAGACAGGA-3' (SEQ ID NO.: 8) for PCR reaction, the PCR product was directly sequenced, or the PCR product was cloned into the pGEM-T vector (purchased from Promega Company) Sequence again.

[0158] result

[0159] Whole-genome exome seque...

Embodiment 2

[0161] Example 2.RNA extraction, RT-PCR and realtimePCR

[0162] In this example, RevertAidTMFirstStrandcDNASynthesisKit (purchased from Fermentas Company of the United States, product number K1622), SYBRGreenRealtimePCRMasterMix (purchased from TOYOBO Company of the United States, product number TY-QPK-201), Taq enzyme and other reagents and instruments were used in this example.

[0163] The target gene primers are as follows:

[0164] BMXΔN (primer sequence used in RT-PCR):

[0165] Forward primer: 5'-AGGGTGGGATTTGATATTGCATGG-3' (SEQ ID NO.:9)

[0166] Reverseprimer: 5'-CCAGGGACACAGAGTCGGGGA-3' (SEQ ID NO.: 10)

[0167] BMXΔN (primer sequence used in realtime):

[0168] Forward primer: 5'-CAGTAACCAAAAAAGAAAGAAATG-3' (SEQ ID NO.: 11)

[0169] Reverseprimer: 5'-TGTGTTGATGATAATGAATAAGC-3' (SEQ ID NO.: 12)

[0170] GAPDH: Forwardprimer: 5'-GCGACACCCACTCCTCCACCTTT-3' (SEQ ID NO.: 13)

[0171] Reverseprimer: 5'-TGCTGTAGCCAAATTCGTTGTCATA-3' (SEQ ID NO.: 14)

[0172] RT-PCR ...

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Abstract

A BMX spliceosome is provided. The nucleotide sequence of the BMX spliceosome is shown as SEQ ID NO:3. A host cell containing a gene coding the BMX spliceosome and a method of detecting functions of the BMX spliceosome by utilization of the host cell are also provided by the invention. The BMX spliceosome or the host cell containing the BMX spliceosome can be used for testing drug resistance of lung cancer cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to BMX splice body and its application in lung cancer drug resistance. Background technique [0002] Lung cancer is one of the most dangerous diseases in the world today. Clinically, although the third-generation platinum-based combination therapy has some advantages in the treatment of non-small cell lung cancer, the combination of several cytotoxic drugs has not achieved better results than platinum double-agent therapy, indicating that this treatment method The bottleneck has been reached. [0003] With the in-depth study of the biology of lung cancer and the further understanding of the molecular mechanism of lung cancer pathogenesis, targeted molecular targeted therapy will be carried out according to the key pathogenic genes in individual tumors of different patients. A relatively successful example is the small molecule inhibitors targeting the kinase domain of EGFR designed fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/11C12N15/63C12Q1/68C12Q1/48
Inventor 季红斌刘红艳汪烨李飞
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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