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Human Pcid2 protein soluble expression method, anti-human Pcid2 protein monoclonal antibody 2D7-F11, and hybridoma cell line secreting antibody

A monoclonal antibody, soluble technology, applied in the direction of anti-animal/human immunoglobulin, analytical materials, animal/human peptides, etc., can solve the problems of decreased number of B cells, apoptosis, etc.

Active Publication Date: 2013-07-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another study pointed out that in Pcid2 conditional knockout mice, specifically knocking out the Pcid2 protein in B cells can lead to apoptosis during the development of B cells, and ultimately lead to a decrease in the number of B cells

Method used

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  • Human Pcid2 protein soluble expression method, anti-human Pcid2 protein monoclonal antibody 2D7-F11, and hybridoma cell line secreting antibody
  • Human Pcid2 protein soluble expression method, anti-human Pcid2 protein monoclonal antibody 2D7-F11, and hybridoma cell line secreting antibody
  • Human Pcid2 protein soluble expression method, anti-human Pcid2 protein monoclonal antibody 2D7-F11, and hybridoma cell line secreting antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation and purification of soluble Pcid2 recombinant protein

[0039] (1) Expression plasmid construction

[0040] Generally speaking, the prokaryotic expression strategy using Escherichia coli is the simplest and easiest protein expression strategy in terms of the difficulty of expressing recombinant proteins. We successfully obtained soluble Pcid2 protein by prokaryotic expression. First, in order to express the Pcid2 protein and its interacting protein Dss1, we amplified the two sequences (NCBI sequence numbers NP_001120675.1, NP_006295.1) from cDNAs using PCR technology, and constructed them into Novagen after restriction enzyme digestion and ligation. The commercialized prokaryotic expression vector pETDuet-1, in which Pcid2 protein has a His tag for purification of recombinant protein, while Dss1 does not have any tag. The pDuet plasmid ensures simultaneous translational expression of both proteins.

[0041] Expression vector construction process:

[0042...

Embodiment 2

[0083] (1) Preparation of hybridoma

[0084] The recombinant human Pcid2 / Dss1 protein obtained in Example 1 was used as an antigen to immunize Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), intraperitoneally injected, and each mouse was injected with 50 micrograms of recombinant human Pcid2 / Dss1 protein. Two weeks later, the mice were immunized again, with the same injection volume and method. After the mouse serum titer reaches the requirement, that is, the titer reaches above 1:200, the cell fusion is prepared, and the mice are subjected to shock immunization three days before the fusion.

[0085] Mouse myeloma cells Sp2 / 0 (ATCC CRL-1772) were prepared at the same time as the mice were immunized.

[0086] Fusion of sensitized B lymphocytes isolated from the spleen of immunized mice with myeloma cells Sp2 / 0 (ATCC CRL-1772) ("Practical Immunology", edited by Yang Tingbin, Changchun Publishing House, published in December 1994) ,...

Embodiment 3

[0093] Identification of 2D7-F11 mAb

[0094] The 2D7-F11 monoclonal antibody prepared in Example 2 was subjected to western blotting on recombinant human Pcid2 protein and human tumor cell line cell lysate according to conventional methods. like figure 2 As shown, the 2D7-F11 monoclonal antibody has a strong positive reaction with recombinant human Pcid2 protein and cells expressing human natural Pcid2 protein, but has no cross-reaction with other proteins including Dss1.

[0095] The 2D7-F11 monoclonal antibody prepared in Example 2 was subjected to immunofluorescence reaction against tumor cell lines HeLa, MCF-7, HepG2 (purchased from Xiehe Cell Bank) and the like according to conventional methods. The results showed that the 2D7-F11 mAb had a strong positive reaction with cells expressing human native Pcid2, as shown in image 3 displayed.

[0096] The 2D7-F11 monoclonal antibody prepared in Example 2 performs immunoprecipitation reaction with the lysate of MCF-7 cells...

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Abstract

The invention provides a human Pcid2 protein soluble expression method, an anti-human Pcid2 protein monoclonal antibody obtained through using a soluble Pcid2 recombinant protein as an antigen of immune mice, and a hybridoma cell line secreting the antibody. The monoclonal antibody has a strong positive reaction to the recombinant human Pcid2 protein and cells expressing a human natural Pcid2 protein, and has no cross reactions to other proteins. The invention also provides a kit including the monoclonal antibody, and a method for detecting the endogenous Pcid2 content through using the monoclonal antibody.

Description

technical field [0001] The present invention relates to the field of protein recombinant expression and monoclonal antibody, more specifically, the present invention relates to the soluble recombinant expression of anti-human Pcid2 protein, and the monoclonal antibody 2D7-F11 prepared by using the soluble expressed Pcid2 protein as an antigen, which is produced by small Produced by the murine hybridoma cell line 2D7-F11. Background technique [0002] Pcid2 (PCI domain containing 2) protein is a highly conserved protein in eukaryotic cells. Its typical structural feature is that there is a PCI (proteasome, COP9 signalosome, initiation factor 3) domain in its sequence. PCI domain is a kind of functional domain widely present in eukaryotic proteasome, COP9 complex and transcription initiation factor 3 complex. Generally located at the C-terminus of related proteins, about 200 amino acids, usually used to mediate protein-protein interactions in protein complexes. Proteins con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/70C07K14/47C07K16/18C12N5/20G01N33/68G01N33/577
Inventor 范祖森钟超李翀杨轩刘朝霞
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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