Preparation, identification and application of HA tag monoclonal antibody

A monoclonal antibody and labeling technology, applied in the field of biomedicine, can solve problems such as buried in the fusion target protein, increased protein purification rate, and protein aggregation

Inactive Publication Date: 2009-09-16
朱晓林
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the purification of HA6-tag fusion protein by this method has many obvious advantages compared with other tag fusion proteins, this method also has its disadvantages: in the presence of metal ions, polyhistidine tends to form a hem-like This is a hydrophobic structure, so after fusion with some proteins, it may be buried inside the fusion target protein; elution with imidazole will lead to protein aggregation
In addition, there is still room for improvement in the purification rate of genetically engineered proteins with HA-tag. Whether the vacancy can be effectively filled by the HA affinity chromatography column with as little loss as possible still needs further experimental exploration

Method used

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  • Preparation, identification and application of HA tag monoclonal antibody

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Embodiment Construction

[0021] 1. Materials:

[0022] HA hexapeptide was synthesized by GenScript Company; ovalbumin (OVA), bovine serum albumin (BSA), rabbit anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM antibodies were purchased from Sigma Company; HRP-labeled goat anti-mouse IgG was provided by Santa Cruz; MULTISKAN MK3 microplate reader was purchased from Thermo; 3326 CO 2 The incubator was purchased from Forma Company; the nitrocellulose membrane (NC membrane) was purchased from Amersham Company; the SDS-PAGE electrophoresis instrument was purchased from BIO-RAD Company; BALB / c pure-line male mice (rats aged 6-8 weeks, body mass 18-22g) was purchased from the Institute of Zoology, Chinese Academy of Medical Sciences.

[0023] 2. Method

[0024] 2.1 Synthesis of artificial antigen

[0025] (1) Synthesis of immune antigen: Weigh 4.4mg HA-tag and 5mg OVA and dissolve in 1mL PB (0.05mol / L, pH7.0). After fully dissolved, add 200μL of PB solution containing 2.2mg EDC dropwise , mixed while drop...

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Abstract

The invention relates to preparation, identification and application of HA tag monoclonal antibody, which belong to the technical field of biological medicines. A carbodiimide method is used for synthesizing an HA-tag complete antigen to immunize a BALB / c mouse, a hybrid tumor method is adopted to perform cell fusion, and positive hybrid tumor cell strains are cloned and screened by a limiting dilution method and an indirect ELISA method. Ascites is prepared conventionally and is purified by an octylic acid-ammonium sulphate method, and specificity identification is performed on a purified mAb. The result of the identification shows that the HA-tag complete antigen is coupled successfully, and a hybrid tumor cell strain excreting an HA tag mAB is obtained successfully through cell fusion, screening and cloning. In the preparation of the ascites, the ascites titer is measured to be more than 1:10<6>, and the strain of the mAb does not have cross reactions with other fusion protein tags; and the monoclonal antibody applied to a Western-blotting and an immunohistochemical detection of fusion proteins so that a method which can be applied to the purification and the detection of HA fusion proteins is established.

Description

technical field [0001] The preparation, identification and application of HA label monoclonal antibody belong to the technical field of biomedicine. Background technique [0002] HA-tag is a short peptide composed of 9 histidines (YPYDVPDYA), specially designed for adsorption and purification of recombinant proteins. Compared with other tags, the HA-tag fusion tag has many obvious advantages, and is currently the most widely used fusion tag for purification. An efficient detection and purification system based on fusion protein can be established by using HA tag. At present, there are not many types of commercialized HA-tag monoclonal antibodies (mAb), and the price is also very expensive. Therefore, the development of HA-tag mAb has very broad application prospects in the study of fusion proteins. [0003] Fusion tags can be divided into two categories according to their relative molecular mass (Mr): large protein molecules (or protein domains and their derivatives) and ...

Claims

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Application Information

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IPC IPC(8): C12N15/06C12P21/08G01N33/577
Inventor 朱晓林
Owner 朱晓林
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