Preparation, identification and application of HA tag monoclonal antibody

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 ...

AI Technical Summary

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

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