DNA aptamer capable of being specifically combined with clostridium difficile toxin TcdB as well as preparation method and application of DNA aptamer

An aptamer, Clostridium pseudobacteria technology, applied in the fields of molecular microbiology and infection immunology, can solve the problems of reports without screening aptamers, and achieve the effects of avoiding drug resistance problems, low screening costs, and alleviating pathological changes.

Active Publication Date: 2022-07-12
HUBEI UNIVERSITY OF MEDICINE
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Problems solved by technology

However, so far, the use of SELEX screening technology to develop oligonucleotide aptamers in the application of anti-clostridium difficile infection has only been reported as a diagnostic reagent (Su Heng et al., International Journal of Laboratory Medicine, 2018, p. Vol. 39, No. 20; Meng Liu e

Method used

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Examples

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Example Embodiment

[0050] Example 1 SELEX screening and obtaining high-affinity aptamers against the FBD region of TcdB

[0051] 1. Construction of random single-stranded DNA library and primers

[0052] Construct a random single-stranded DNA library with a length of 88 bases, as shown in SEQ ID No.6: 5'-GCG GAATTC TAATACGACTCACTATAGGGAACAGTCCGAGCC-N30-GGGTCAATGCGTCATA-3', where N represents four random bases of A, T, C, and G. The upstream primer shown in SEQ ID No.1 is: 5'-GCG GAATTC TAATACGACTCACTATAGGGAACAGTCCGAGCC-3', the underlined part is the DNA restriction enzyme cleavage site of EcoR I; the downstream primer shown in SEQ ID No.2 is: 5'-GCG GGATCC TATGACGCATTGACCC-3', the underlined part is the DNA restriction enzyme cleavage site of Bam HI, random single-stranded DNA (ssDNA) library and primers can be synthesized by the company.

[0053] Nucleic acid sequence table 1

[0054]

[0055] 2. PCR amplification and preservation of double-stranded DNA (dsDNA) and single-stranded DNA (s

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PUM

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Abstract

The invention discloses a DNA aptamer capable of being specifically combined with clostridium difficile toxin TcdB and a preparation method and application thereof.The nucleotide sequence of the DNA aptamer is as shown in SEQ ID No.4. The DNA aptamer is obtained through screening with the SELEX technology, the screening process is simple, the screening cost is low, the screening time is short, the screened aptamer is cloned to a pUC19 vector, and the DNA aptamer can be used for detecting clostridium difficile toxin TcdB. And the aptamer is transformed into escherichia coli DH5alpha, so that a large number of aptamers can be directly prepared through the strain. The aptamer can be specifically combined with TcdB, the toxic effect of the aptamer on intestinal epithelium is reduced, local macrophages of intestinal tissue are inhibited from secreting IL-1beta and IL-6, damage of inflammatory reaction to the intestinal tissue is relieved, and therefore infection of clostridium difficile is effectively treated.

Description

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Claims

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Application Information

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Owner HUBEI UNIVERSITY OF MEDICINE
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