Application of leucocythemia related to gene ASH2L in leukemia molecule classification diagnosis

A technology for molecular typing and leukemia, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of uncertain expression characteristics of ASH2L, limitations of ASH2L in molecular typing diagnosis, unclear expression characteristics, etc.

Inactive Publication Date: 2006-11-29
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the absence of an in-depth understanding of the mechanism of action of ASH2L, it remains unclear whether functionally related molecules of ASH2L have similar expression characteristics during PMA-stimula...

Method used

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  • Application of leucocythemia related to gene ASH2L in leukemia molecule classification diagnosis
  • Application of leucocythemia related to gene ASH2L in leukemia molecule classification diagnosis
  • Application of leucocythemia related to gene ASH2L in leukemia molecule classification diagnosis

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Embodiment 1

[0025] Primer name

Embodiment 2

[0027] Cloning of ASH2L, DPY-30-like and WDR5 genes

[0028] K562 cells were cultured on a 10cm dish with R1640 containing 10% FBS at 37 degrees, 5% CO 2 Cultured until the number of cells reached 2×10 7 Above, cells were harvested to extract total RNA and subjected to reverse transcription. Reverse transcription was carried out using a kit from PROMEGA, and the method is shown in the kit instructions. Using the cDNA as a template, three genes ASH2L, DPY-30-like and WDR5 were amplified. The primers used were ASH2L-1 / 2, DPY-1 / 2 and WDR5-1 / 2 (see Example 1 for the sequence). The conditions of the PCR reaction are: ASH2L, annealing temperature 64 degrees, extension 7 minutes; DPY-30-like, annealing temperature 60 degrees, extension 1 minute; WDR5 annealing temperature 60 degrees, extension 1.5 minutes; other conditions are the same Common PCR conditions, using Pfu enzyme.

[0029]After the PCR product was tailed with Taq enzyme at 72 degrees for 10 minutes, it was directly c...

Embodiment 3

[0031] Preparation of anti-ASH2L, DPY-30-like specific antibodies:

[0032] Antigen preparation: ASH2L and DPY-30-like cDNA template PCR amplification required fragments, primers are ASH2L-6P-UP / ASH2L-6P-DOWN and DPY-6P-UP / DPY-6P-DOWN two Right (see Example 1 for the sequence). The target fragment on ASH2L is the N-terminal 1-99aa coding region, and DPY-30-like is its full length. Both ends of the PCR fragment contain restriction enzyme sites EcoR I and Xho I, so these two enzymes were used to digest the PCR product and vector plasmid pGEX-6P-1. Then use T4 ligase to ligate the digested PCR fragment and the vector to form a recombinant plasmid (see figure 1 ).

[0033] PCR conditions: the annealing temperature is 65 degrees, the extension time is 1 minute, and other conditions are the same as ordinary PCR reactions. PCR reaction product 50ul (approximately 5ug) and vector plasmid (pGEX-6P-1) 6-10ug were digested with EcoR I and Xho I respectively, and DNA fragments were re...

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Abstract

The ASH2L expression would be specific decrease in the PMA exciting K562 differentiation process. And the DPY-30-like and WDR5 have no such alteration. Thus, ASH2L could be used as the alternate mark material to diagnose leukaemia. It constructs the N-end albumen of ASH2L and DPY-30-like full length albumen plasmid to transform to bacilluscoli expression corresponding albumen. It could be used to diagnose bacilluscoli diagnose.

Description

technical field [0001] The invention belongs to the fields of gene recombination, antibody preparation and clinical diagnosis. Specifically, the present invention relates to a recombinant genetic engineering and protein purification process of the N-terminal part of the leukemia-related gene ASH2L, to the recombinant genetic engineering and protein purification process of the full-length ASH2L interacting protein DPY-30-like, and to the use of The preparation of highly specific antibodies from these two purified proteins involves the specific downregulation of ASH2L during the differentiation process of K562 cells stimulated by PMA, the recombinant plasmids containing part of the ASH2L gene and the recombinant strains expressing the protein and their use in the molecular typing diagnosis of leukemia Applications. Background technique [0002] Accurate classification of leukemia is an important prerequisite for the treatment of leukemia. At present, the diagnosis and typing...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N1/21C12N15/70C07K16/18G01N33/53C12Q1/68C12N15/09
Inventor 彭勇汪俊华袁建刚彭小忠强伯勤
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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