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B lymphocyte stimulator dodecamer as well as preparation method and application thereof

A B lymphocyte and stimulating factor technology, applied in the fields of biotechnology and immunology, can solve the problems of low detection window, poor sensitivity and low binding ability, and achieve the effect of large detection window, high sensitivity and good experimental repeatability.

Active Publication Date: 2021-03-26
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although BLyS exists in a trimer form in its native state, its ability to bind receptors is not high, resulting in a low detection window and poor sensitivity in these assays

Method used

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  • B lymphocyte stimulator dodecamer as well as preparation method and application thereof
  • B lymphocyte stimulator dodecamer as well as preparation method and application thereof
  • B lymphocyte stimulator dodecamer as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of biotinylated BLyS trimer protein

[0048] 1. Experimental materials

[0049] TransT1 competent cells (full gold, Cat. No. CD501-01); tryptone (Sigma, Cat. No. T7293-1KG), yeast extract (Sigma, Cat. No. Y1625-1KG); agar (Sigma , Cat. No. A7002-1KG); Max plasmad Kit (Promega, Cat. No. A2492); Bacterial shaker (Uno, HN211B); Bacterial incubator (Tianjin Test Instrument Co., Ltd., DH3600B) Nanodrop 2000; Expi293F cells (Invitrogen, Cat.No.R790-07); cell expression medium (CD 293 TGE Medium, 1×, Acrobiosystems, Cat. No. CM-1156-11-1L); pcDNA3.1 plasmid (ThermoFisher, Cat No. V79520); Polyethyleneimine, PEI (Polysciences, Cat. No. 23966). Cell shaker incubator (Uno, HNY-2102). Nickel column (NI SEPHAROSEHP, Cat. No. GE, 17-5268-01).

[0050] 2. Solution preparation

[0051] LB medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, add water to 1000ml, autoclave, cool to room temperature, add Amp, the final concentration is 100ug / ml, set asi...

Embodiment 2

[0076] Example 2 Preparation of BLyS protein dodecamer

[0077] 1. Experimental materials

[0078] Biotinylated BLyS trimer protein; Streptavidin (Streptavidin, SA, Sigma, Cat. No.S0677-1MG); Phosphate buffered saline (PBS buffer, Hyclone, Cat. No. SH30256.01) .

[0079] 2. Preparation of BLyS protein dodecamer

[0080]Prepare 1mL biotinylated BLyS trimeric protein solution with PBS buffer solution, the concentration is 400ug / ml. Prepare 1mL Streptavidin solution with PBS buffer solution, the concentration is 100ug / ml. Add the prepared Streptavidin solution to the B lymphocyte stimulating factor protein solution in 5 times, add 200uL each time, place it on a horizontal shaker to react at room temperature, the interval of addition is 10min, and place it on a horizontal shaker after adding the last 200uL Continue to react for 30min. The reaction molar ratio of B lymphocyte stimulating factor protein to Streptavidin is 4:1; BLyS protein dodecamer is obtained; it is packed in...

Embodiment 3

[0081] Example 3 Identification of dodecamer activity of BLyS protein

[0082] 1. Experimental materials

[0083] Mouse spleen cells (ALLCELLS, Cat. No. MSPL-001F); human CD19+ B cells (TPCS, Cat. No. CD19-N-Custom); fetal bovine serum (Fetal bovine serum, CellMax, Cat. No. SA201 .02); RPMI1640 medium (RPMI1640 medium, Hyclone, Cat. No. SH30809.01); lipopolysaccharide (Lipopolysaccharide, LPS, Sigma-Aldrich, Cat. No. L6529); anti-IgM antibody (Invitrogen, Cat. No. 16-5099-85; Biotinylated BLyS protein (Biotinylated Human BAFF Protein, HisAvitag™ active trimer, Acrobiosystems, Cat. No. BAF-H82Q2); BLyS protein (HumanBAFF Protein active trimer, Acrobiosystems, Cat. No. BAF- H52D4); CellTiter-Glo® Luminescent Cell Viability Assay Kit (CellTiter-Glo® Luminescent Cell Viability Assay, Promage, Cat. No. G7572).

[0084] 2. Stimulate mouse spleen cell proliferation

[0085] Prepare RPMI1640 complete medium containing 10% fetal bovine serum and 5ug / mL lipopolysaccharide; resuscitat...

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Abstract

The invention provides a B lymphocyte stimulator dodecamer as well as a preparation method and application thereof. The dodecamer is obtained by coupling a biotinylated B lymphocyte stimulator trimerprotein and streptavidin according to a molar ratio of 4: 1. The B lymphocyte stimulator dodecamer provided by the invention shows biological activity obviously superior to that of a BLyS protein in mouse spleen cell proliferation stimulating experiments and human B cell proliferation experiments, and has the characteristics of large detection window, high sensitivity, good experiment repeatability and the like when being used for screening BLyS-related neutralizing antibodies.

Description

technical field [0001] The invention relates to the fields of biotechnology and immunology, in particular to a dodecamer of B lymphocyte stimulating factor and its preparation method and application. Background technique [0002] B lymphocyte stimulator (BLyS / B-cell activating factor, BAFF) is a member of the tumor necrosis factor superfamily, mainly expressed in myeloid family cells including monocytes, macrophages, dendritic cells, granulocytes, malignant B cells, etc. B-lymphocyte-stimulating factors are expressed in the form of trimers on the surface of these cell membranes, and then released into the microcirculation after being cleaved by Furin protease, and then bind to receptors on the surface of B-cell membranes. BLyS mainly has three receptors, transmembrane activator and CAML Interactor (transmembrane Activator and CAML Interactor, TACI), B cell maturation antigen (B cell maturation antigen, BCMA) and B cell-activating factor receptor (B cell-activating factor re...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85C07K16/28C07K1/14G01N33/68
CPCC07K14/70575C07K16/2875C07K2317/76C07K2319/21C07K2319/40C12N15/85C12N2800/107G01N33/6854G01N2333/70575
Inventor 石晓娟陈宜顶苗景赟刘丛张敬晗
Owner ACROBIOSYSTEMS INC
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