Diagnostic reagent and kit for cancer

A kit and cancer technology, applied in the medical field, can solve the problem of not finding the correlation between hmc and cancer

Inactive Publication Date: 2013-05-08
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current prior art, no correlation between hmc and cancer has been found

Method used

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  • Diagnostic reagent and kit for cancer
  • Diagnostic reagent and kit for cancer
  • Diagnostic reagent and kit for cancer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] The preparation of embodiment 1,5-hydroxymethylcytosine (hmc) antigen

[0090] 5-Hydroxymethylcytosine (hmc) is a small molecular compound, and direct injection into the body of rabbits, rats, mice or other immunized animals is not enough to cause antigen-antibody immune response. Therefore, the inventors coupled hmc as a hapten to carrier proteins such as BSA (bovine serum albumin), OVA (ovalbumin), KLH (hemocyanin), and then injected it into rabbits, mice, etc. The polyclonal antibody that recognizes hmc is prepared in immunized animals, and the monoclonal antibody that recognizes hmc is prepared by fusion with cloned cells and screening.

[0091] 1. Method and product detection of bovine serum albumin BSA coupled with 5-hydroxymethylcytosine hmc

[0092] (1) Weigh 10mg hmc (PY7596, Berry & Associates, Inc.USA), and put it into a 1.5ml Ep tube;

[0093] (2) Add 500μl 0.1M NaIO 4 , react with hmc in the dark at room temperature for 20min;

[0094] (3) Add 30 μl of ...

Embodiment 2

[0131] The preparation method of embodiment 2, 5-hydroxymethylcytosine (hmc) antibody

[0132] Preparation of rabbit polyclonal antibody

[0133] The inventor injected hmc coupled to BSA carrier protein into rabbits to obtain immune serum. The preparation method is as follows: hmc-coupled bovine serum albumin (BSA) is mixed with Freund's adjuvant, injected into rabbits, immunized with 0.5 mg, immunized once every 2 weeks, and immunized three times. The rabbit polyclonal antibody against hmC was obtained after routine affinity purification.

[0134] Western blot experiments showed that the rabbit polyclonal antibody HMC-PA01 only recognized hmc, but basically did not recognize 5-methylcytosine (5mC) ( Figure 5 ) and other bases such as adenine, guanine, cytosine, and thymine.

[0135] Preparation of mouse monoclonal antibodies

[0136] The present inventors injected hmc coupled to BSA carrier protein into mice, obtained immune serum, selected mice producing hmc antibodies ...

Embodiment 3

[0139] Experimental application of embodiment 3, hmc antibody

[0140] 1. Rabbit polyclonal antibody can specifically detect 5-hydroxymethylcytosine hmc

[0141] In order to detect whether the HMC-PA01 polyclonal antibody can be used for immunostaining, the inventors used RT-PCR to obtain the cDNA of the enzyme Tet1 that catalyzes the production of hmc from mouse embryonic stem cells. The coding sequence of amino acids (based on the protein sequence of GenBank accession number: NM030625), with Nhe I and Bam HI restriction sites set at both ends. After digestion with NheI and BamHI, pIRES2-EGFP containing the coding sequence of green fluorescent protein (GFP) was inserted to construct a plasmid pmTET1-ires-GFP, and it was transferred into 293T cells (CRL-11268, ATCC, USA). GFP was used to distinguish the transferred and non-transferred cells, and the transferred cells were positive for GFP, on the contrary, the non-transferred cells were negative for GFP. These cells were imm...

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Abstract

The present invention relates to a diagnostic reagent and a kit for cancer. The present invention discloses that the existence condition of 5-hydroxymethylcytosine (hmc) in organizations is closely related to the incidence and development of cancer, the significant decrease of the hmc level in the organization indicates incidence or development of cancer, and also the hmc level indicates the severity of cancer. Therefore, the hmc detection reagent can be well applied to cancer diagnosis, prognosis or parting, and has a high clinical application value.

Description

technical field [0001] The invention belongs to the medical field; more specifically, the invention relates to a reagent and a kit for diagnosing cancer. Background technique [0002] DNA methylation is a common epigenetic modification. In mammals, DNA methylation mainly refers to the carbon at the 5th position of cytosine (C) being methylated and modified into 5-methylcytosine (5mC) . CpG dinucleotides are the main sites of DNA methylation, which are unevenly distributed in the genome. In some regions, mainly the gene promoter and the first exon region, the CpG probability is higher than normal, and these regions are therefore called CpG islands. In the entire genome, about 60% of the promoter regions of genes contain CpG islands. In general, 5mC accounts for about 2-7% of the total C in mammals, so it is called the "fifth base". [0003] DNA methylation plays an important role in normal and abnormal physiological activities. It can inhibit gene expression, participate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/544C07K14/765C07K14/77C07K14/795C07K16/44C12N5/20C12R1/91
Inventor 陈德桂张丰刘一帆
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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