Recombination cyclophilin A antibody, preparation method thereof, enzyme-linked immuno sorbent assay (ELISA) kit and cell strains

Abstract

The invention relates to a recombination cyclophilin A antibody, a preparation method thereof, an enzyme-linked immuno sorbent assay (ELISA) kit and cell strains. The problems that process conditions are complex, the specificity is poor, and protein immunological activity loss is large in the process of preparing the existing recombination cyclophilin A antibody are solved. Induced bacteria whichare purified or not purified immunize experiment animals, and a specific polyclonal antibody or a specific monoclonal antibody of the recombination cyclophilin A can be obtained through screening andpurification. The recombination cyclophilin A antibody is prepared through the preparation method. A solid phase carrier and a detection antibody in the ELISA kit contain the recombination cyclophilin A antibodies. The preparation method of the recombination cyclophilin A antibody has the advantages that the process conditions are simple, the specificity is good, the purification is simple, the protein immunological activity loss is less, and the operation is convenient.

Description

technical field [0001] The invention relates to the field of molecular biology and immunology, in particular to a recombinant human cyclophilin A antibody, its preparation method, ELISA kit and cell line. Background technique [0002] There are more than 130 isomers of cyclophilins that have been discovered and cloned. They constitute the Cyclophilins family, which are widely distributed in bacteria, fungi, plants and mammals, and are relatively conservative in the evolution process. Cyclophilin was originally discovered as a protein receptor of cyclosporin A (Cyclosporin A, CsA), which is an immunosuppressant and is clinically used to treat rejection and autoimmunity in organ transplantation Systemic diseases, so cyclophilins are thought to be involved in the immunosuppressive process. Later, a large number of studies have shown that cyclophilin also has many other important biological functions. CyPs has peptide prolyl cis-trans isomerase (PPIase) activity, which catalyze...

AI Technical Summary

Problems solved by technology

[0005] For the detection of CypA protein, the immunological method is undoubtedly the most convenient one. If the immunological method is used for detection, it is necessary to first establish a detection method and prepare a kit. The core component of the kit - the preparation of antibodies. The traditional method is: To produce antibodies with recombinant proteins, the general process is: first culture and induce the expression bacteria, collect and lyse the induced expression bacteria, after the tagged recombinant protein is purified by the tag, it is necessary to use protease to excise the carrier fusion tag protein, and the untagged recombinant protein The protein is purified according to the protein characteristics. If the expressed protein is in the inclusion body, then the inclusion body purification method is carried out. Protein purification is still a difficult problem in the field of biology. It needs the comprehensive use of many methods. Not only that, but often the purity of the protein and The solubility is not high, and the biological activity is more difficult to guarantee; if the antibody is prepared, it is necessary to use the above purified protein to immunize animal experiments, and finally use the purified protein as an affinity ligand to purify the antibody

The procedure is cumbersome, the cost of protease is high, and it is not suitable for large-scale industrial production, and each additional step will not only lose at least 20% of the protein amount (Table 2), but also lose about 30% of the protein immunological activity (Table 3); if The recombinant protein is expressed in the inclusion body, and the purification of the recombinant protein is even more difficult; since there are many kinds of proteins in the bacteria, any method of purification will inevitably encounter bacteria proteins similar in structure or properties to the target protein. The protein-like protein is difficult to remove during the purification process, resulting in low purity of the purified protein, and sometimes it is difficult to see clearly by running SDS-PAGE gel. Use this protein (as an antigen) to immunize animals and use it as an affinity purification antigen partner Some of the antibodies produced by purifying antibodies or screening monoclonal antibody cell lines are bound to target E. coli bacterial proteins remaining in the purified protein (antigen), and people are exposed to E. coli every day in life, and there are also E. coli in the body. Some antibodies against Escherichia coli bacterial protein, if the actual kit produced by the above method is used to detect human specimens, there is no doubt that it will definitely interfere with the real results, resulting in a decline in kit performance or even ineffectiveness

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