A dna aptamer capable of specifically binding bcg glycolipid antigen and its application

A specific and aptamer technology, applied in the fields of molecular microbiology and infection immunology, to achieve the effect of enhancing cell response, improving immunogenicity and low cost

Active Publication Date: 2014-10-22
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no report on the use of aptamers screened by SELEX screening technology in anti-tuberculosis vaccines as an adjuvant to enhance BCG immunity

Method used

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  • A dna aptamer capable of specifically binding bcg glycolipid antigen and its application
  • A dna aptamer capable of specifically binding bcg glycolipid antigen and its application
  • A dna aptamer capable of specifically binding bcg glycolipid antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Screening of DNA Adapters of Purified BCG ManLAM

[0035] The present invention has the screening method of the DNA aptamer that specifically binds to BCG as follows:

[0036] 1.1 Construction of random single-stranded DNA library and primers

[0037] To construct a single-stranded DNA library of 88 bases in length:

[0038] 5'-GCGGAATTCTAATACGACTCACTATAGGGAACAGTCCGAGCC-N30-GGGTCAATGCGTCATA-3', where N stands for A, T, C, G four random bases;

[0039] The upstream primer is: 5'-GCG GAATTC TAATACGACTCACTATAGGGAACAGTCCGAGCC-3’, the underlined part is Eco DNA restriction enzyme site for RI;

[0040] The downstream primer is: 5'-GCG GGATCC TATGACGCATTGACCC-3', the underlined part is Bam DNA restriction enzyme site for HI.

[0041] Random single-stranded DNA libraries and primers can be synthesized by the company.

[0042] 1.2 PCR amplification and storage of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) libraries

[0043] Before each rou...

Embodiment 2

[0051] Example 2 Screening of DNA Adapter BM2 with High Affinity to BCG

[0052] Ask the company to synthesize the aptamer BM2 with fluorescent FAM label, and then use flow cytometry to measure the affinity with different bacteria.

[0053] (1) Take 100μL 1×10 7 CFU of BCG, H37Rv and Mycobacterium smegmatis and other bacteria were placed in EP tubes, fixed with 75% ethanol for 15 minutes, and washed 3 times with PBS.

[0054] (3) Add 4 μg of fluorescently labeled gamete BM2 to each tube, mix well, and incubate at 37°C for 2 hours.

[0055] (4) Wash with PBS for 3 times, add 500 μL of PBS, and perform upstream flow detection.

[0056] The result is as figure 2 , the aptamer BM2 has the highest affinity with BCG, and has a very low affinity with other bacteria such as mycobacteria and Staphylococcus aureus, indicating that the aptamer BM2 has strong specificity for binding BCG and can block its surface immunosuppressive molecule ManLAM Immunosuppressive effect, has the val...

Embodiment 3

[0057] Example 3 The mechanism of BM2 as an adjuvant to improve the immunogenicity of BCG

[0058] The screened ssDNA aptamer can specifically bind to ManLAM on the surface of BCG, greatly reducing its immunosuppressive effect, recruiting and activating more dendritic cells to peripheral lymph nodes, inhibiting regulatory T cells (Tregs) (CD25+FoxP3+), Regulatory B cells (Bregs) (CD19+IL-10+) proliferate, enhance Th1 cell responses, etc., thereby improving the immunogenicity of existing BCG. The specific implementation methods are as follows:

[0059] (1) 30 male BALB / c mice (age: 6-8 weeks, purchased from Hubei Provincial Center for Disease Control and Prevention) were randomly divided into 5 groups, with 6 mice in each group. See Table 1 for detailed experimental groups:

[0060] Table 1. Grouping and immunization routes of BALB / c mice

[0061] Experimental group immune pathway Dose / only only count 1. PBS subcutaneous injection 100 μL 6 2. B...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) aptamer capable of specifically bonding with a BCG (bacillus Calmette-Gue'rin) glycolipid antigen and an application of the DNA aptamer, belonging to the fields of molecular microbiology and immunology of infection. The nucleotide sequence of the DNA aptamer is as shown by SEQ ID No.1, and the DNA aptamer can be specifically bonded with BCG surface ManLAM (Mannosylated lipoarabinomannan) to collect and activate more dendritic cells to peripheral lymph glands, inhibit the multiplication of adjustable T cells and adjustable B cells, enhance reaction and the like of Th1 type cells and improve the immunogenicity of BCG; and a mouse tuberculosis toxic material eliminating experience shows that after the BCG and the DNA aptamer are used for immunizing a mouse together, an immune protection effect which is stronger than that of single BCG immunization is obtained. The DNA aptamer disclosed by the invention can be used for preparing a novel immunologic adjuvant to enhance the immunogenicity of BCG anti-tuberculosis infection, and provides a new strategy and a new vaccine adjuvant for preventing and treating tuberculosis.

Description

technical field [0001] The invention belongs to the fields of molecular microbiology and infection immunology, and relates to a DNA aptamer capable of specifically binding BCG glycolipid antigen and its application. Background technique [0002] Tuberculosis (Tuberculosis, TB) is a widespread and harmful infectious disease. It is also known as the world's three major infectious diseases together with AIDS and malaria. One-third of the world's people have been infected with tuberculosis, and more than 1.7 million people die from tuberculosis every year. It consumes a lot of manpower and material resources to prevent and control TB. [0003] Currently, the only widely used vaccine approved for the prevention of tuberculosis is BCG (bacillus Calmette-Gue′rin). Years of practice have shown that BCG only has a certain protective effect on children, and the protective effect on adults varies greatly, with its preventive effect ranging from 0% to 80%. The effectiveness of this tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115A61K39/39A61P31/06
Inventor 章晓联孙小明王其龙
Owner WUHAN UNIV
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