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IRAK-M polyclonal antibody and preparation method thereof

A technology of polyclonal antibody and antigen, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, anti-enzyme immunoglobulin, etc., can solve problems such as lack, and achieve good antigenicity, single molecular antigenicity, and molecular reaction sex good effect

Active Publication Date: 2019-03-22
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide an IRAK-M multi- cloned antibody

Method used

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  • IRAK-M polyclonal antibody and preparation method thereof
  • IRAK-M polyclonal antibody and preparation method thereof
  • IRAK-M polyclonal antibody and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] The preparation of embodiment 1 immunogen and IRAK-M polyclonal antibody

[0030] Use forward primer F: 5'-cga ggatcc ctcgaacatcagagctctacc-3' with reverse primer R: 5'-GCG aagctt ggcaacacttgctttcttccc-3' used the plasmid vector containing the porcine IRAK-M open reading frame as a template to amplify the IRAK-M gene fragment by PCR. The list is shown in SEQ ID NO.2. The fragment amplified by PCR was double-digested with BamHI and HindIII, and connected into the pET28a vector to construct the pET-IRAK-M prokaryotic expression vector plasmid. The recombinant protein predicted to be expressed has 289 amino acids, and its sequence is as shown in SEQ ID NO. 1 with the 6His tag.

[0031] Express and purify the recombinant protein to obtain the immunogen. The specific steps are as follows:

[0032] 1) Transform the pET-IRAK-M prokaryotic expression plasmid into Escherichia coli BL21 competent cells, culture in LB liquid medium at 50 μg / mL kanamycin at 200 rpm at 37°C, a...

Embodiment 2 Embodiment 1

[0038] Example 2 The IRAK-M polyclonal antibody prepared in Example 1 was detected by Western hybridization to detect the eukaryotic expression of IRAK-M-3×Flag fusion protein in CHO cells

[0039] Western detection steps are as follows:

[0040] 1) Transfect CHO cells with an overexpression vector expressing IRAK-M fused with a 3×Flag tag at the N-terminus, and collect the cell pellet in a centrifuge tube after 24 hours;

[0041] 2) Add cell lysate to Nonidet P-40 (NP-40) buffer (1% SDS, 1% NP-40, 50mM Tris (pH8.0), 150mM NaCl, 4mM Pefabloc SC, 2mg / ml Leupeptin, 2mg / ml aprotinin), boiled for 5min after becoming clear, centrifuged at 12000rpm at 4°C for 10min, and the supernatant was taken;

[0042] 3) Add 1 / 4 volume of 5× protein loading buffer (2% SDS, 10% glycerol, 60mM Tris (pH6.8), 5% β-mercaptoethanol and 0.01% bromophenol blue) to the supernatant, boil for 10min, 10 After %SDS-PAGE gel 120V electrophoresis for 90min, 240mA was used to transfer the PVDF membrane for 2h...

Embodiment 3I

[0044] Example 3 Immune Reaction of IRAK-M Polyclonal Antibody with IRAK-M in Human and Porcine Cells

[0045] The MARC145 cells used in this example are monkey embryonic kidney epithelial cells, the HeLa cells are human cervical cancer cells, the A549 cells are human lung adenocarcinoma cells, and the 3D4 cells are immortalized porcine alveolar macrophages.

[0046] Cell lysis, SDS PAGE electrophoresis and Western operation were the same as in Example 2, wherein the primary antibody used was the IRAK-M polyclonal antibody prepared in Example 1.

[0047] The result is as figure 2 As shown, the IRAK-M polyclonal antibody of the present invention did not react with IRAK-M in the lysate of monkey cells MARC145 and human cells HeLa and A549, but specifically reacted with IRAK-M in the lysate of pig cell 3D4 reaction.

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Abstract

The invention provides an IRAK-M antigen, nucleic acid for coding a protein of which an amino acid sequence is shown as a sequence table SEQ ID NO:1, a carrier containing the nucleic acid for coding the protein of which the amino acid sequence is shown as the sequence table SEQ ID NO:1, a transformant containing the carrier, as well as an IRAK-M polyclonal antibody and a preparation method thereof. The IRAK-M polyclonal antibody is prepared by the preparation method including the following steps: taking the protein of which the amino acid sequence is shown as the sequence table SEQ ID NO:1 asan immunogen to immunize an animal, and obtaining the IRAK-M polyclonal antibody from the animal. The IRAK-M polyclonal antibody has high reactivity with pig IRAK-M molecules, does not undergo any non-specific reaction which has an influence on target protein detection in Western hybridization, and can specifically perform a good immunoreaction with pig IRAK-M in a protein Western blotting and fluorescence detection test.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to an IRAK-M polyclonal antibody and a preparation method thereof. Background technique [0002] IRAK-M (also known as IRAK-3) is a newly discovered molecule in the IRAK family. A series of molecules in the IRAK family play an important role in the transmission of signals mediated by TLRs (Toll-like Receptors) and IL-1Rs (Interleukin-1 receptor) IRAK-M is the only negative regulatory factor found in this family, and the animal model of IRAK-M deficiency shows enhanced TLR signal, that is, IRAK-M can inhibit TLR signal transduction. In addition, IRAK-M was also found to be involved in negative regulation of immunity in models of bacterial infection or endotoxin tolerance. [0003] The existing sources of IRAK-M protein include mice and humans. According to the records in the NCBI database, there is a predicted sequence (ACCESSION Number XM_003126363) of the porcine IRAK-M molecul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/63C07K16/40G01N33/573
CPCG01N33/573C12N9/1205C07K16/40G01N2333/9121
Inventor 邱亚峰马志永杜翔魏建超刘珂李蓓蓓邵东华
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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