Prokaryotic Expression Of The Human IgE Receptor FcεRIα-D2and Its Selectivity Adsorption Capacity For IgE

Type I allergy, known as allergic reactions, is a kind of immunological disease caused by excessive IgE. Removal of excessive IgE from the blood of patients suffering from allergic reactions is greatly meaningful for relieving or curing allergic disease. Medication is the main treatment for allergic disease and exhibits good therapeutic effects, but it is unable to cure allergic disease and will lead to drug dependence. As the intractable allergies is caused by the long-term existence of sensibiligen specific IgE, selective removal of IgE from patients’blood by blood purification could significantly relieve the allergy. Existing adsorbents with the heterologous proteins as the ligands show low capacity of selective adsorbing IgE. Besides, the detachment of the ligands from the matrix will trigger immune response in human body. In order to improve the safety and the specific adsorption capacity of the adsorbent, we prepared the adsorbent for human IgE by using human IgE recetor, FcεRIa, as the ligand, and investigated its adsorption properties. The contents of this study are listed below:(1) Recombinant plasmids pET28a-FcεRIa-D2,pET23a-FcεRIa-D2, pCW-ori-FcεRIa-D2and pFLAG-ATS-FcεRIa-D2were constructed by genetic engineering, and the expressions of recombinant plasmids in same host were compared with each other. E.coli BL21(DE3)-pET23a-FcεRIa-D2was selected as expressing strain, fort its expression amount of FcεRIa-D2was the highest in the four candidates and could be up to30%of total protein. Finally the optimal culture condition was defined as an overnight activation followed by1%inoculattion to LB medium for5h, and then the induction with0.2mM IPTG for4h. All steps were operated at37°c.(2) The recombinant protein was expressed as inclusion bodies. Two methods were applied to renature the target protein: SEC purification after the renaturation by dialysis reached54.5%renatured protein. Protein purification and renaturation were conducted at the same time with strong anion exchange chromatography, which finally made44.9%of inclusion bodies renatued. The purity of the recombinant protein purified from both methods was higher than95%, but the renatured protein from dialysis process showed better adsorption capacity for IgE. Thus, dialysis was chosen for the following renaturation. The renatured protein was water soluble with the molecular weight of12.9kDa. (3) The adsorbent was prepared by immobilizing the recombinant FcεRIα-D2on Sepharose CL-4B, and its adsorption capacity for serum IgE was investigated by static adsorption. The adsorbent with a ligand density of0.4-4mg/mL was able to remove about30%of IgE from the serum of patients suffering from allergic reactions. These results showed that the recombinant FcεRIα-D2could significantly and selectively remove IgE from the patients’ serum.Our study demonstrated that the recombinant human IgE receptor, FcεRIα-D2, could be applied as a specific ligand for preparing the adsorbent. This novel adsorbent was able to specifically remove IgE, which would be very promising for the cure of allergic reactions.