Objective To prepare the recombinant protein of the group 33 allergen of Dermatophagoides pteronyssinus (Der p 33), and to determine its serum IgE-binding rate. Methods The primers were designed according to the published sequence of Der p 33 (GenBank No. AUX14773), and the Der p 33 gene of D. pteronyssinus was amplified by RT-PCR. After being identified by agarose gel electrophoresis, the amplification product was connected to the pET-28a (+) vector and heat-transformed into Escherichia coli, and the positive recombinant plasmid was selected for sequencing. The pET-28a (+)-Der p 33 plasmid was transformed into E. coli Rosetta 2 (DE3) plysS, expressed with the induction by isopropyl-β-D-thiogalactoside (IPTG), and purified by column chromatography and was then subjected to Western blotting analysis. The structure and physicochemical properties of Der p 33 were predicted by bioinformatics software. Results The encoding gene of Der p 33 was obtained, which had a full length of 1 383 bp and a relative molecular weight of 51 432.81 and encoded 460 amino acids. The secondary structure was predicted to contain α-helix (40.87%), β-turn (7.17%), extended strand (17.39%), and random coil (34.57%). The purity of recombinant protein was >95.00%. According to the Western blotting analysis, the serum IgE-binding rate of recombinant Der p 33 was 18.75% (3/16). Conclusion The Der p 33 gene was successfully cloned, and the purified recombinant protein had serum IgE-binding activity, which lays a foundation for the development of diagnostic reagents for the allergen component of D. pteronyssinus and the bioproducts of desensitization treatment.
Li YANG, Nan WANG, Fei-xiang TENG, Gui-fang MA, Jin-xia SUN
. Preparation of the recombinant protein of Der p 33 from Dermatophagoides pteronyssinus and determination of its serum IgE-binding rate[J]. Chinese Journal of Vector Biology and Control, 2023
, 34(1)
: 21
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DOI: 10.11853/j.issn.1003.8280.2023.01.004
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