Nano antibody molecule targeting dendritic cells and application

A dendritic cell and nanobody technology, applied in the field of biomedicine, can solve problems such as differences in the intensity of immune responses, and achieve the effects of low autoimmunity, small molecular weight, and accelerated processing

Active Publication Date: 2021-09-24
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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  • Nano antibody molecule targeting dendritic cells and application
  • Nano antibody molecule targeting dendritic cells and application
  • Nano antibody molecule targeting dendritic cells and 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 total RNA extraction

[0034] Vacuum anticoagulant tubes were used to collect 5 mL of peripheral blood from each of 6 alpacas, and shake gently to mix the blood and anticoagulant thoroughly. The fresh blood was brought back to the laboratory according to the operating instructions of the Tianjin Haoyang Peripheral Blood Lymphocyte Separation Kit. The obtained lymphocytes were washed with 10mL cell washing solution, centrifuged at 250×g for 10 minutes, the supernatant was discarded, and the washing was repeated twice. The cells were then resuspended with 2 mL of PBS buffer for use. Total RNA of lymphocytes was extracted according to the operating instructions of Solebol Total RNA Extraction Kit (R1200). The total RNA was used as a template, and two primers, Random 6 and Oligo dT (Bao Biology), were used to reverse transcribe into cDNA. Th...

Embodiment 2

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

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

[0044] SPF chicks about 15 days old were killed by injecting air needles, soaked in 75% ethanol for 10 minutes, took out the chicken femur and tibia under aseptic conditions, washed out the bone marrow with PBS containing 2% double antibody, collected bone marrow cells, 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 the 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 liquid level layer, PBS Resuspend and wash twice, centrifuge at 150×g for 10 minutes each time. Collect the cell pellet, resuspend the cells with 2 mL of 1640 medium containing 10% FBS and 1% doubl...

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 the Nanobody library to 1 × 10 11 PFU / mL, chicken bone marrow cells (5×10 5 cells / mL) were mixed, incubated at 37°C for 30 minutes, centrifuged at 150×g for 10 minutes, and the supernatant was collected and marked as Input. The chicken dendritic cells (1 × 10 6 cells), incubated at 37°C for 30 minutes. Centrifuge at 150×g for 10 minutes, remove the supernatant, and mark it as Unbound. The cells were resuspended in 1640 culture medium containing 0.05% Tween-20 and 10% FBS, centrifuged at 150×g for 10 minutes, the supernatant was removed, marked as Wash 1, and the washing operation was repeated 5 t...

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Abstract

The invention provides a nano antibody molecule targeting dendritic cells and application. The amino acid sequences of the nano antibody molecule targeting the dendritic cells are as shown in SEQ ID NO. 1 and SEQ ID NO. 2. An alpaca-derived natural nano antibody T7 phage display library is constructed, and nano antibody molecules which can be combined with chicken bone marrow DCs in a high-affinity manner are screened by using an affinity screening technology. Specificity detection shows that the two screened nano antibody molecules not only can be combined with the DCs of chickens, but also can be combined with DCs of poultry such as ducks and geese, but are not combined with bone marrow mononuclear cells and fibroblasts of chickens (ducks and geese) except the DCs, and have better DCs combination specificity. When the nano antibody molecule is used as a subunit antigen carrier, the immunoreaction excited by the antigen molecule can be remarkably improved, and the antibody level is improved. Therefore, the nano antibody molecule screened by the invention can be used as a targeting carrier of an antigen molecule, the antigen presentation efficiency is improved, and a support is provided for the development of a novel poultry vaccine.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, relates to a nanobody molecule targeting dendritic cells and its application, in particular to the screening of the nanobody molecule and the preparation of vaccines using the nanobody molecule as an antigen carrier. Background technique [0002] Dendritic cells (DCs) are a type of antigen-presenting cells (Antigen presenting cells, APCs) in the innate immune system of mammals, which mainly perform the functions of capturing, processing and presenting antigens. Once activated, DCs will migrate to lymph nodes and combine with T cells and B cells here to activate the adaptive immune system. At the same time, DC can also interact with cells of the innate immune system such as natural killer cells, phagocytes and mast cells, and is a bridge connecting innate immunity and adaptive immunity. DCs are considered to be the most effective antigen-presenting cells that can activate initial and memory i...

Claims

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Application Information

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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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