Phosphorylated EIF5B specific antibody and preparation method and application thereof

A specificity and phosphorylation technology, applied in chemical instruments and methods, antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of poor specificity and few types of EIF5B antibody antibodies

Active Publication Date: 2021-05-18
南昌大学第一附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For this reason, the technical problem to be solved by the present invention is that there are few types and poor specificity of EIF5B ant...

Method used

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  • Phosphorylated EIF5B specific antibody and preparation method and application thereof
  • Phosphorylated EIF5B specific antibody and preparation method and application thereof
  • Phosphorylated EIF5B specific antibody and preparation method and application thereof

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Experimental program
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Embodiment

[0034] This embodiment provides a phosphorylated EIF5B-specific antibody, specifically, the antibody is a polyclonal antibody specific to human phosphorylated EIF5BT78.

[0035] Phosphorylated ElF5B T78 specific site polyclonal antibody was prepared by the following method:

[0036] S1. Prepare the antigen. First, synthesize 20 mg of polypeptide 1 according to the protein sequence of EIF5B (as shown in SEQ ID NO: 3). The amino acid sequence of the polypeptide 1 is as shown in SEQ ID NO: 1: KADRET(p)VAVKPTENN-Cys, In SEQ ID NO: 1, threochloric acid (T) at this site is phosphorylated. Take 10 mg of polypeptide 1 coupled with hemocyanin (KLH) to prepare the antigen. The mass spectrometry results of polypeptide 1 and the polypeptide coupled with KLH are as follows: Figure 1-2 shown.

[0037] S2. Prepare an antibody, and immunize the polypeptide 1 coupled with hemocyanin as an antigen to obtain an antibody. Two New Zealand white rabbits were immunized with KLH-coupled polypept...

experiment example

[0109] 1. Antibody endogenous verification

[0110] Western Blot (WB) verification:

[0111] The main instruments used are shown in Table 2 below:

[0112] Table 2

[0113]

[0114] The main reagents used are shown in Table 3 below:

[0115] table 3

[0116]

[0117] The detection process specifically includes the following steps:

[0118] Sample preparation: Use A549 and 143B cells to prepare phosphorylated EIF5B activated cell samples, collect the cells, use RIPA lysate ((RIPA Lysis Buffer)) to lyse the cells at 4°C, and lyse the cells at 4°C at a speed of 12000rpm Centrifuge for 20min and transfer the supernatant to a new EP tube. The protein concentration was determined by BCA method, and 5×SDS loading buffer was added according to the volume of the lysate, and boiled at 100°C for 5 minutes. To prevent protein degradation, operations should be performed on ice as much as possible.

[0119] SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis): clamp th...

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Abstract

The invention discloses a phosphorylated EIF5B specific antibody and a preparation method and application thereof. The antibody is a human-derived phosphorylated ElF5BT78 specific site polyclonal antibody. On one hand, the antibody provides possibility for detecting expression of a phosphorylated EIF5BT78 site and verifying a related molecular mechanism; and on the other hand, the antibody is combined with phosphorylated eukaryotic translation initiation factor 5B (p-EIF5B) protein. The invention further provides a composition containing the antibody. The specific phosphorylated EIF5BT78 site polyclonal antibody is constructed by utilizing an existing phosphorylated protein antibody technology, the problems that in the prior art, EIF5B phosphorylated antibodies are few in variety and poor in specificity are solved, and possibility is provided for detecting expression of the phosphorylated EIF5BT78 site and verifying the related molecular mechanism.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a phosphorylated EIF5B specific antibody and its preparation method and application. Background technique [0002] Eukaryotic translation initiation factor 5B (eukaryotic translation initiation factors, eIF5B), also known as translation initiation factor IF-2, is located on chromosome 2 and plays an important role in translation initiation. Pairs with the promoter-tRNA and codon at the P site to initiate protein translation. Meanwhile, EIF5B is also required for specific mRNA translation, helping to support or stabilize Met-tRNA binding. Earlier studies found that EIF5B delivered Met-tRNA to the P site of the ribosome to initiate viral translation in the IRES-dependent translation initiation mechanism of CSFV (swine fever virus) and HCV (hepatitis C virus). [0003] EIF5B is highly expressed in a variety of tumors, and plays a decisive role in the pathophysiology of can...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K16/06A61K39/395A61P35/00
CPCC07K16/30C07K16/065A61P35/00A61K2039/505
Inventor 刘志礼吴昕刘家明
Owner 南昌大学第一附属医院
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