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CD81 aptamer and application thereof

An aptamer and nucleic acid aptamer technology, applied in the biological field, can solve the problems of exosome purity, integrity and process maturity, high price, and time-consuming

Active Publication Date: 2021-05-28
SUZHOU GENEPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the exosomes obtained by these methods are not perfect in terms of purity, integrity and process maturity, and the disadvantages of time-consuming and high price are unavoidable

Method used

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  • CD81 aptamer and application thereof
  • CD81 aptamer and application thereof
  • CD81 aptamer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1. Screening of aptamers targeting CD81

[0072] The aptamer screening of CD81 is based on the SELEX technology of CD81 recombinant protein and CD81 positive expression cell line (the schematic diagram is shown in figure 1 shown), the specific way is: in about 10 14 Aptamers targeting CD81 were screened within a library of aptamer-ssDNA. The DNA in the library all contains the following core sequence (86nt, wherein N is any other base): 5'-TAG GGA AGAGAA GGA CAT ATG AT-40N-TTG ACT AGT ACA TGA CCA CTT GA-3'. The library contains 671 DNA sequences shown in Table 12. Aptamers bound to CD81 recombinant protein or CD81-positive cell lines were eluted and amplified by PCR. The sequences of the upstream and downstream primers for PCR amplification are: FITC-5'-TA GGG AAG AGAAGG ACA TAT GAT-3' and 5'-TTT TTT TTT TTT TTT TTT TT / iSp9 / T CAA GTG GTC ATG TACTAG TCA A- 3'. The amplified DNA was identified by high-throughput sequencing, and 6 aptamers with the best bindin...

Embodiment 2

[0075] Example 2. Identification of binding ability of CD81 aptamer and CD81 at cell level

[0076] 1. Construction of recombinant animal cells overexpressing CD81

[0077] The specific construction method of the recombinant animal cell (the cell is named 293T / CD81) overexpressed CD81 is as follows: the KpnI and XbaI of the plasmid pCMV3-C-His (Beijing Yiqiao Shenzhou Technology Co., Ltd., the product of Sino Biological Inc.) The sequence between the recognition sites is replaced by the cDNA sequence of human CD81, and the recombinant expression vector that keeps other parts of the pCMV3-C-His sequence unchanged, the recombinant expression vector is named pCMV3-CD81. pCMV3-CD81 was introduced into HEK293T cells to obtain recombinant cells, which were named 293T / CD81. The nucleotide sequence of the cDNA of human CD81 is the nucleotide sequence shown in 7 in the sequence table, and its encoded amino acid sequence is the protein with his tag shown in the sequence.

[0078] 2. T...

Embodiment 3

[0090] Example 3. Identification of binding ability of CD81-2J and CD81-2F subtypes

[0091] For CD81-2J and CD81-2F, we constructed 8 subclones respectively, and the sequences are shown in Table 5 and Table 6. By the test method in Example 2, compare the binding force of the subtypes of CD81-2J and CD81-2F as follows: Figure 8-11 shown. The results demonstrated that subtypes CD81-2J-1, CD81-2J-6 and CD81-2F-2 had significantly higher binding ability to CD81-overexpressing HEK293T cells (293T / CD81) than negative control HepG2 cells (without CD81) (* *p<0.1, ***p<0.01), indicating that the above three subtypes have stronger binding ability to CD81 in its natural conformation. Therefore, CD81-2J-1, CD81-2J-6 and CD81-2F-2 were selected for subsequent verification and detection experiments.

[0092] Table 5 Sequences of 8 subtypes of aptamer CD81-2J

[0093]

[0094]

[0095] Table 6 Sequences of 8 subtypes of aptamer CD81-2J

[0096]

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Abstract

The invention relates to the technical field of biology, in particular to a CD81 aptamer and application thereof. The CD81 aptamer has (a) a nucleotide sequence as shown in a sequence 1 or (b) a sequence obtained by deleting or replacing one or more bases of the nucleotide sequence as shown in the sequence 1. The combination of the CD81 aptamer provided by the invention and the CD81 has extremely high selectivity and specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an aptamer of CD81 and its application. Background technique [0002] An aptamer is a small single-stranded oligonucleotide sequence (DNA / RNA) obtained through screening, which can bind with a corresponding ligand with high affinity and strong specificity. Aptamers can bind to macromolecules such as proteins and cells; they can also bind to a small segment of polypeptides, small molecule drugs, sugar compounds and ions. Aptamers can be rapidly synthesized, identified, and modified, and have the advantages of high repeatability, high purity, and low price, making aptamers play an important role in early diagnosis, monitoring, and treatment of diseases, especially exosome-based diagnosis. [0003] Exosomes are a type of membrane vesicle structure (30-150nm) containing small RNA, DNA and protein. Exosomes are widely found in biological fluids, including blood, saliva, uri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/574G01N33/569G01N33/543
CPCC12N15/115G01N33/68G01N33/57415G01N33/57419G01N33/57438G01N33/57423G01N33/57492G01N33/56966G01N33/54326C12N2310/16G01N2333/70596G01N33/569G01N33/543G01N33/574
Inventor 段维张佩琢
Owner SUZHOU GENEPHARMA
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