A nucleic acid aptamer, kit and method for detecting pancreatic ductal carcinoma

A technology of nucleic acid aptamer and pancreatic ductal carcinoma, which is applied in the fields of biochemical equipment and methods, measurement/testing of microorganisms, DNA preparation, etc., which can solve the problems of lack of sensitivity of pancreatic cancer, long antibody preparation cycle, and difficult immunogenicity, etc. problem, to achieve non-immunogenicity, short cycle, high affinity and specificity

Active Publication Date: 2017-07-28
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, on the one hand, antibodies have many defects such as long preparation period, high cost, easy inactivation, immunogenicity and difficult chemical modification; on the other hand, clinical tumor markers of pancreatic cancer (such as carbohydrate antigen CA19-9, CA-50 and carcinoembryonic antigen) lack specificity
In addition, the above tumor markers also lack sensitivity to early pancreatic cancer.
These issues limit the application of antibody-based targeted molecular probes in the early diagnosis and treatment of pancreatic cancer

Method used

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  • A nucleic acid aptamer, kit and method for detecting pancreatic ductal carcinoma
  • A nucleic acid aptamer, kit and method for detecting pancreatic ductal carcinoma
  • A nucleic acid aptamer, kit and method for detecting pancreatic ductal carcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Screening of pancreatic ductal carcinoma cell-specific nucleic acid aptamers

[0039] (1) Design of the nucleic acid library and primers used:

[0040] Random ssDNA library:

[0041] 5'-ACCGACCGTGCTGGACTCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTATGAGCGAGCCTGGCG-3' (N represents A, T, C and G four bases) (SEQ ID NO.3), that is, the theoretical library capacity of the random single-stranded DNA library is 4 42 DNA;

[0042] Upstream primer: 5'-fluorescein isothiocyanate-ACCGACCGTGCTGGACTCA-3' (SEQ ID NO.4);

[0043] Downstream primer: 5'-biotin-CGCCAGGCTCGCTCATAGT-3' (SEQ ID NO.5);

[0044] (2) Positive screening:

[0045] 2.1 Incubation: Dissolve the above random nucleic acid library with binding buffer (D-PBS, 5mM magnesium chloride), denature at a constant temperature of 95°C for 5 minutes, and quickly put it on ice; then mix it with the cultured and pretreated pancreatic ductal carcinoma cell line PL45 Incubate for 1 hour at 4°C.

[0046] 2.2 Dis...

Embodiment 2

[0057] Example 2: Screening of pancreatic ductal carcinoma cell-specific nucleic acid aptamers

[0058] The obtained nucleic acid aptamers were labeled with fluorescent molecules at the 5' end to make molecular probes, and the molecular probes of 0nM, 20nM, 40nM, 60nM, 80nM, 100nM, 120nM, 140nM, 160nM, 200nM, 300nM, 400nM, 500nM were taken respectively. needle solution, with 3 x 10 5 PL45 cells were incubated on ice for 1 hour, then washed twice with washing buffer, and the fluorescence intensity on the cell surface was measured by flow cytometry. If the cells can bind fluorescently labeled nucleic acid aptamers, plot the fluorescence intensity against the probe concentration, and use the formula Y=BmaxX / (Kd+X) to calculate the equilibrium dissociation constant Kd of the nucleic acid aptamers. The results showed that the equilibrium dissociation constants of the nucleic acid aptamers were all in the nanomolar range.

Embodiment 3

[0059] Embodiment 3: Analysis of the stability of nucleic acid aptamer (SEQ ID NO.2) in complete medium

[0060] 3 micromolar fluorescein isothiocyanate-labeled nucleic acid aptamer (SEQ ID NO.2) was respectively placed in 200 microliters of DMEM cell culture medium containing 10% fetal calf serum. At different investigation time points, the samples were quickly cooled in dry ice-ethanol and then stored in a -80°C refrigerator. When the samples at all time points were collected, the samples were placed at 4° C., and after they were dissolved, 20 microliters of samples were taken for separation in 4% agarose gel, and then imaging analysis was performed. Such as Figure 4 It can be seen that the nucleic acid aptamer (SEQ ID NO.2) can exist stably in the above medium for 6 hours.

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Abstract

The invention provides an aptamer, a kit and a method for detecting pancreatic duct adenocarcinoma. The sequences of the aptamer are as shown in SEQ ID NO.1 and SEQ ID NO.2. The aptamer capable of identifying the pancreatic duct adenocarcinoma cell line PL45 is capable of identifying the pancreatic duct adenocarcinoma cell line PL45 specifically and with high affinity, and is highly in binding capacity. The danger of tumors can be layered molecularly by use of the aptamer; the aptamer is capable of specifically recognizing and binding tumor metastatic foci and identifying targets, and then a good method can be provided for diagnosing and treating the metastatic foci, and the method plays an important role in early detection of the pancreatic duct adenocarcinoma.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid aptamer, a kit and a method for detecting pancreatic ductal carcinoma. Background technique [0002] Pancreatic cancer (Pancreatic carcinoma) is a cancer that occurs in the pancreas, which can occur in the head, body, tail and the entire pancreas. Pancreatic cancer is pathologically classified into pancreatic ductal adenocarcinoma, carcinoma of special ductal origin, acinar cell carcinoma, small glandular carcinoma, large eosinophilic granulosa cell carcinoma, and small cell carcinoma, among which pancreatic ductal adenocarcinoma accounts for the 80-90% of cancer cases. In recent years, the incidence of pancreatic cancer has shown an obvious upward trend in my country. According to <<2012 China Tumor Registration Annual Report>> statistics, the incidence of pancreatic cancer has ranked ninth among all malignant tumors in my country. At presen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/04
Inventor 谭蔚泓赵子龙武晓秋叶茂白华荣
Owner HUNAN UNIV
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