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A nanobody molecule targeting dendritic cells and its application

A technology of dendritic cells and nano-antibodies, which is applied in the field of biomedicine, can solve problems such as differences in the intensity of immune responses, and achieve the effects of low autoimmunogenicity, small molecular weight, and accelerated processing

Active Publication Date: 2022-05-31
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many advantages in developing vaccines by directly targeting antigens to DCs, researchers still face many difficulties and challenges
The receptors targeted by different ligands are different, and the ligands targeting the same receptor have differences in their binding sites, targeting properties, and affinities, which lead to differences in the intensity of the activated immune response

Method used

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  • A nanobody molecule targeting dendritic cells and its application
  • A nanobody molecule targeting dendritic cells and its application
  • A nanobody molecule targeting dendritic cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of Alpaca Nanobody T7 Phage Display Library

[0033] 1. Isolation of alpaca peripheral blood lymphocytes and extraction of total RNA

[0034] Collect 5 mL of peripheral blood from 6 alpacas in a vacuum anticoagulant tube, shake gently to mix blood and anticoagulant thoroughly. Bring fresh blood back to the laboratory and operate according to the operating instructions of Tianjin Haoyang Peripheral Blood Lymphocyte Separation Kit. The obtained lymphocytes are washed with 10 mL of cell washing solution, centrifuged at 250 × g for 10 minutes, the supernatant is discarded, and the washing is repeated twice. The cells were then resuspended in 2 mL of PBS buffer for use. According to the operating instructions of Solebao Total RNA Extraction Kit (R1200), the total RNA of lymphocyte cells was extracted. Using total RNA as a template, reverse transcription was performed into cDNA with two primers, Random 6 and Oligo dT (Bao Bio). The reaction program w...

Embodiment 2

[0042] Example 2 Isolation, culture and identification of chicken bone marrow-derived dendritic cells

[0043] 1. Isolation and culture of chicken bone marrow-derived cells

[0044] SPF chicks around 15 days of age were killed by air needle injection and then immersed in 75% ethanol for 10 min. The chicken femur and tibia were taken out under sterile conditions, and the bone marrow was washed out with PBS containing 2% double antibody, and bone marrow cells were collected. PBS buffer Wash twice, each time at 150 × g, centrifuge for 10 minutes, and discard the supernatant. Resuspend the pelleted cells in PBS, then slowly add the bone marrow cells to a centrifuge tube containing Histopaque-1119 (Sigma) according to the volume ratio of 1:1, centrifuge at 150×g for 30 minutes, collect the white cell layer at the layer of the liquid surface, PBS Resuspend and wash twice at 150 x g for 10 min each. The cell pellet was collected, and the cells were resuspended in 2 mL of 1640 mediu...

Embodiment 3

[0049] Example 3 Affinity Panning of Alpaca Nanobody T7 Phage Display Library

[0050] 1. Subtractive screening of chicken dendritic cells targeting nanobody molecules

[0051] The chicken dendritic cells prepared in Example 2 were subjected to three rounds of affinity panning using the alpaca nanobody T7 phage display library constructed in Example 1. Adjust the concentration of Nanobody library to 1×10 11 PFU / mL, chicken bone marrow cells (5 × 10) resuspended with 2 mL of 1640 medium containing 10% FBS 5 cells / mL), incubate at 37°C for 30 minutes, centrifuge at 150 × g for 10 minutes, collect the supernatant and mark it as Input. Use the collected supernatant to resuspend the chicken dendritic cells cultured in Example 2 to the 8th day (1 × 10 6 cells), incubated at 37°C for 30 minutes. Centrifuge at 150 x g for 10 minutes, remove the supernatant and label as Unbound. The cells were resuspended in 1640 medium containing 0.05% Tween-20 and 10% FBS, centrifuged at 150 × g...

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Abstract

The invention provides a nanobody molecule targeting dendritic cells and its application. The amino acid sequences of the dendritic cell-targeting nanobody molecules are shown in SEQ ID NO.1 and SEQ ID NO.2. The invention constructs alpaca-derived natural nanobody T7 phage display library, and utilizes an affinity screening technique to pan out nanobody molecules capable of binding to chicken bone marrow DCs with high affinity. Specificity detection showed that the two nanobody molecules screened not only had the ability to bind to chicken DCs, but also to duck, goose and other poultry DCs, and did not bind to bone marrow mononuclear cells other than DCs, chicken (duck, goose) Fibroblast binding, with better DCs binding specificity. Using the nanobody molecule as a subunit antigen carrier can significantly improve the immune response stimulated by the antigen molecule and increase the antibody level. Therefore, the nanobody molecules screened by the present invention can be used as targeting carriers of antigen molecules, improve the efficiency of antigen presentation, and provide support for the development of new poultry vaccines.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a nanobody molecule targeting dendritic cells and its application, in particular to the screening of nanobody molecules and the preparation of vaccines by using the nanobody molecule as an antigen carrier. Background technique [0002] Dendritic cells (DCs) are a type of antigen presenting cells (APCs) in the mammalian innate immune system, which mainly play the functions of antigen capture, processing and presentation. Once activated, DCs will metastasize to lymph nodes, where they combine with T cells and B cells to activate the adaptive immune system. At the same time, DCs can also interact with cells of the innate immune system, such as natural killer cells, phagocytes, and mast cells, and act as a bridge between innate immunity and adaptive immunity. DCs are considered to be the most effective antigen-presenting cells capable of activating primary and memory immune respons...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18A61K39/385A61P37/04
CPCC07K16/18C07K16/005A61K39/385A61P37/04C07K2317/22C07K2317/569A61K2039/6056Y02A50/30
Inventor 徐海李玲李睿婷郭子杰洪伟鸣朱善元
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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