BAFF receptor (BCMA), an immunoregulatory agent
A technology for immunoglobulin, autoimmune disease, applied in the field of β-cell activating factor
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Embodiment 1
[0138] Embodiment 1: Detect the combination of BAFF and BAFF-R with plate test
[0139] This Example describes the assay of BAFF binding to BAFF-R transfected cells using a plate assay.
[0140] The full-length human BAFF-R was prepared from BJAB polyA+RNA using the Superscript II preamplification kit (Life Technologies) to obtain a cDNA template, and primers complementary to the 5' and 3' end coding sequences of BAFF-R were used, Amplification was performed with Pful. The resulting PCR product was cloned into CH269, which was derived from pCEP4 (Invitrogen). The resulting clone was named pJST535. Human embryonic kidney cells (293EBNA) containing the EBNA-1 gene were inoculated into 6-well culture plates coated with fibronectin, and then passed lipofectamine with different concentrations of pJST535 and CH269 as background control (lipofectamine) (Life Technologies) for transfection. 48 hours after transfection, the ability of transfected cells to bind soluble flag-hBAFF (a...
Embodiment 2
[0142] Example 2: Determination of the binding of BAFF to BAFF-R transfected cells using a fluorescence activated cell sorter (FACS)
[0143] This example describes the detection of BAFF binding to BAFF-R transfected cells using a FACS assay.
[0144] Plasmid pJST535 encoding full-length BAFF-R was transfected into 293EBNA cells with FuGene6 (Boehringer Mannheim). At 24 hours or 48 hours after transfection, cells were removed from the plate with 5 mM EDTA in PBS and counted. Wash the cells with FACS buffer (PBS containing 10% fetal bovine serum, 0.1% NaN3 and 10 μg / ml hIgG (Jackson ImmunoResearch), and then take 2.5×10 5 Cells were incubated with flag-hBAFF diluted in FACS buffer at a concentration of 9 μg / ml to 0.037 μg / ml and incubated on ice for 1 hour. The above-mentioned cells were washed with FACS buffer, then placed on ice, and incubated with 5 μg / ml anti-FLAG monoclonal antibody M2 for 30 minutes. Wash the cells with FACS buffer, then place the cells on ice, and was...
Embodiment 3
[0146] Example 3: After the introduction of GFP markers, the interaction between BAFF / BAFF-R was determined by FACS
[0147] In this example, the binding of BAFF to cells co-transfected with BAFF-R and GFP reporter plasmids is described.
[0148] According to the method described in Example 2, 293EBNA cells were transfected with pJST535 and GFP reporter plasmids. The reporter plasmid encodes a membrane-anchored GFP molecule. Co-transfected with these two plasmids, we then analyzed the percentage of transfected cells capable of binding BAFF. The cells were taken out from the plate, combined with BAFF, and detected by the method described in Example 2. Also, to exclude dead cells, 7-AAD was introduced. Samples were analyzed by FACS and curves were drawn. The upper right quadrant represents those cells that incorporate BAFF (phycoerythrin positive) and express GFP.
[0149] Although not all GFP-transfected cells were able to bind BAFF, a significantly greater proportion of c...
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