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Nucleic acid aptamer for identifying intestinal cancer serum marker and screening method and application of nucleic acid aptamer

A nucleic acid aptamer and marker technology, used in biochemical equipment and methods, pharmaceutical formulations, and microbial determination/inspection, etc., can solve the problems of complex aptamer screening process and low application value.

Inactive Publication Date: 2019-09-20
YANSHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early aptamers were mostly screened with a single protein as the target, but the application value of single protein screening in serum diagnosis and screening is not high. Therefore, in recent years, the use of multiple targets to screen aptamers has attracted widespread attention.
The difference between compound target screening and conventional screening is that the target is no longer a single protein, but a complex variety of proteins, which makes the whole aptamer screening process more complicated

Method used

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  • Nucleic acid aptamer for identifying intestinal cancer serum marker and screening method and application of nucleic acid aptamer
  • Nucleic acid aptamer for identifying intestinal cancer serum marker and screening method and application of nucleic acid aptamer
  • Nucleic acid aptamer for identifying intestinal cancer serum marker and screening method and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The screening of embodiment 1 nucleic acid aptamer sequence:

[0081] 1. Blood sample processing

[0082] Collect blood samples from 20 first-time patients (not receiving treatment), centrifuge at 3000rpm for 10min at room temperature to remove blood cells, and collect serum; then centrifuge at 15000×g for 30min at 4°C to remove sediment and surface oil, and collect the middle supernatant. Store at -80°C for later use. The blood samples of 20 cases of healthy people were processed in the same way as above.

[0083] 2. Acetonitrile precipitation method to remove high-abundance proteins in serum

[0084] The serum of the above 20 patients with intestinal cancer was mixed thoroughly, prepared according to serum: water: acetonitrile = 1:2:0.5, ultrasonicated in a water bath for 5 min, 15000×g, centrifuged for 30 min, the supernatant was collected, and the precipitate was discarded. Serum from healthy subjects was treated the same as above.

[0085] 3. Synthetic primary ...

Embodiment 2

[0112] Example 2 Nucleic acid aptamer secondary structure prediction:

[0113] The RNA structure 5.7 software was used to predict the secondary structure of the four nucleic acid aptamers obtained from the above screening. The secondary structure prediction results of the four nucleic acid aptamers are as follows: Figure 4 shown.

Embodiment 3 4

[0114] Example 3 Affinity determination between four nucleic acid aptamers and targets:

[0115] Incubate overnight with 8 mL of treated colon cancer serum and 2 mL of carboxy-sepharose magnetic beads, wash with PBS three times, and centrifuge to divide into 40 parts of magnetic bead-serum protein complexes. Dilute the synthesized FAM-labeled 4 nucleic acid aptamers to 0nM, 1nM, 2nM, 5nM, 10nM, 20nM, 50nM, 100nM, 200nM and 500nM, respectively, and then incubate with the magnetic bead-serum complex respectively to prepare mark. Wash 5 times with 1×TPBS, 1 time with ultrapure water, and remove the supernatant. Add 200 μL ultrapure water to resuspend the magnetic beads, and measure the fluorescence value by flow cytometry. Taking the fluorescence intensity as the ordinate and the aptamer concentration as the abscissa, the curve was fitted according to the formula Y=(Bmax×X) / (Kd+X) to obtain the dissociation curves of the four nucleic acid aptamers and calculate the Kd value. T...

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Abstract

The invention relates to a nucleic acid aptamer for identifying an intestinal cancer serum marker and a preparation method and application of the nucleic acid aptamer. The invention specifically discloses nucleotide sequences of four nucleic acid aptamers and a screening method thereof. First, serum samples of firstly diagnosed patients with intestinal cancer and healthy people are collected, primers and ssDNA of a primary library are synthesized, and magnetic agarose microspheres coupled with carboxyl on the surface are used as a serum protein adsorption carrier, and a subtractive SELEX technology and a real-time fluorescence PCR technology are used to obtain gradually enriched nucleic acid aptamer sequences which specifically bind to intestinal cancer serum proteins. Four aptamers for specifically identifying intestinal cancer serum are screened by high throughput sequencing and data analysis. The four nucleic acid aptamers provided by the invention have the characteristics of high affinity and strong specificity for the serum of patients with intestinal cancer, and have wide application prospects in the aspects of research on intestinal cancer tumor marker fishing, initial screening of patients with intestinal cancer and intestinal cancer targeted drug treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid aptamer for recognizing intestinal cancer serum markers, a screening method and application thereof. Background technique [0002] Intestinal cancer is a common malignant tumor that seriously threatens human health. Its incidence rate ranks third after lung cancer and gastric cancer, and it poses a huge threat to people's lives and property. For intestinal cancer, the main reason for threatening human health is that it is discovered late and lacks effective early detection methods for high-risk groups. If high-risk groups can be identified in a painless way like routine physical examinations, treatment methods can be provided early and their survival rates can be improved. At present, the detection of tumor markers is an important means to realize the early diagnosis of intestinal cancer, but there is still a lack of effective and specific intestinal cance...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/6806C12Q1/6869G01N33/574G01N33/53A61K31/711A61P35/00
CPCA61K31/711A61P35/00C12N15/115C12N2310/16C12Q1/6806C12Q1/6869G01N33/5308G01N33/57446C12Q2531/113C12Q2535/122
Inventor 栗坤齐力轻石明李健刘志伟
Owner YANSHAN UNIV
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