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Application of nucleic acid aptamer

A nucleic acid aptamer and Mycoplasma hyorhinosum technology, applied in the field of tumor microbiology, can solve the problems of eukaryotic cell side effects, toxicity, lack of specificity, etc., and achieve good application prospects

Active Publication Date: 2021-09-07
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, mycoplasma detection has not been listed as a diagnostic item for tumor screening in clinical work. More mycoplasma detection is applied to in vitro cell culture. It is very common, and it still has certain side effects and toxicity to eukaryotic cells, lack of specificity, and the killing of normal flora in the human body is also an inherent defect

Method used

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  • Application of nucleic acid aptamer
  • Application of nucleic acid aptamer
  • Application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 constructs the mycoplasma hyorhinois infection cell model

[0083] Cell source: Eca109 cells (esophageal cancer cells) used in this experiment came from the Cell Bank of Wuhan Chinese Academy of Sciences. In the screening process, Eca109 cells infected with mycoplasma (Eca109-mycoplasma infected) were used as forward screening, and uninfected Eca109 cells were used as reverse screening.

[0084] Mycoplasma hyorhinosum (ATCC 17981) was purchased from ATCC in the United States, cultured in Hayflick medium for 5 days until the color of the medium changed from orange-red to yellow, and the concentration of mycoplasma was detected by the gradient dilution method to be 10 6 CCU / ml, culture Eca109 cells to 80% density, dilute the Mycoplasma hyorhini bacteria solution 10 times with the cell culture medium and co-cultivate the cells, subculture after 2 days and add the mycoplasma solution again, repeat this process and stop the cells after subculture 2 times Add my...

Embodiment 2

[0085] Example 2 Cell-SELEX technology for screening nucleic acid aptamers.

[0086] Design nucleic acid library:

[0087] 5'-ACCGACCGTGCTGGACTCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTATGAGCGAGCCTGGCG-3' (N stands for A, T, C, G four bases); SEQ NO.8

[0088] Forward primer: 5'-FITC-ACCGACCGTGCTGGACTCA-3'SEQ NO.9

[0089] Reverse primer: 5'-biotin-CGCCAGGCTCGCTCATAGT-3'SEQ NO.10

[0090] screening process

[0091] In the present invention, Eca109-mycoplasma infected cells infected by Mycoplasma hyorhini are used as positive screening cells, and uninfected Eca109 cells are used as negative screening cells.

[0092] 1. Positive screening:

[0093] a. Dissolve the above library with binding buffer, denature at 95°C for 5 minutes, immediately place it on ice for renaturation for 10 minutes, then remove the culture medium of Eca109-mycoplasma infected cells, add the library solution, and incubate at 4°C for 1 hour. Finally, remove the supernatant solution, wash the cells w...

Embodiment 3

[0101] Example 3 Detection of the binding ability of the nucleic acid sequence obtained by high-throughput sequencing to cells by flow cytometry The nucleic acid sequence with the highest enrichment rate obtained by high-throughput sequencing was named SEQ NO.1, and its sequence is:

[0102] 5'-ACCGACCGTGCTGGACTCACGTCGTCCATTTCCTTGAAAAAGGCACGGGTTCCATGAACTCACTATGAGCGAGCCTGGCG-3';

[0103] Perform base replacement in the middle segment of SEQ NO.1 to obtain a nucleic acid aptamer with the same function: SEQ NO.2:

[0104] 5'-ACCGACCGTGCTGGACTCAAGCGGTTTCCGTGAACCTGCGAGTTCCCTGAATACTTGTACTATGAGCGAGCCTGGCG-3'

[0105] SEQ NO.3:

[0106] 5'-ACCGACCGTGCTGGACTCAGATCCATTTCCTTGAAAAAGGCACGGGTTCCCAGAACTCAACTATGAGCGAGCCTGGCG-3'

[0107] SEQ NO.4:

[0108] 5'-ACCGACCGTGCTGGACTCAATCGGGATCCATGTATCCAGGTTCGTTCCTTGAATGGCACACTATGAGCGAGCCTGGCG-3'

[0109] SEQ NO.5:

[0110] 5'-ACCGACCGTGCTGGACTCAACTGCTGAGCCAATTCTTAATAAAGCACGGGTTCCTAGAACTCAACTATGAGCGAGCCTGGCG-3'

[0111] SEQ NO.6:

[0112] 5'-A...

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PUM

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Abstract

The invention discloses application of a nucleic acid aptamer specifically combined with mycoplasma hyorhinis. The aptamer is high in specificity and sensitivity, can be combined with mycoplasma hyorhinis in a targeted manner, particularly can be combined with p37 protein of mycoplasma hyorhinis in a targeted manner, can also block infection of mycoplasma hyorhinis to cells, inhibits migration and / or invasion of mycoplasma hyorhinis infected cells, treats cells infected by mycoplasma hyorhinis, and increases drug sensitivity of mycoplasma hyorhinis infected cells to gemcitabine. The invention provides a novel method and way for detection, diagnosis and treatment of tumors.

Description

technical field [0001] The invention belongs to the technical field of tumor microbiology, and relates to the preparation of p37 protein targeting and binding to Mycoplasma hyorhina; blocking the infection of cells by Mycoplasma hyorhina; inhibiting the migration and / or invasion of cells infected by Mycoplasma hyorhina; and treating cells infected by Mycoplasma hyorhina; Application of various preparations to increase the drug sensitivity of Mycoplasma hyorhini-infected cells to gemcitabine. Background technique [0002] Malignant tumor is a kind of malignant disease in which normal cells in a living body undergo excessive proliferation, metastasis, and infiltration under the influence of many internal and external factors, thereby endangering the normal organ function of the human body and leading to death. Tumor occurrence factors include the genetic factors of the host as well as external physical, chemical and microbial inducements. According to existing research report...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K47/26C12N15/115A61P31/04
CPCA61K31/7088A61K47/26C12N15/115A61P31/04C12N2310/16
Inventor 叶茂谭蔚泓张毅彬
Owner HUNAN UNIV
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