Cloning, expression, purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus

doi:10.12140/j.issn.1000-7423.2020.06.008

Abstract

Abstract:

Objective To clone the gene of the tenth-class allergen Der p 10 (tropomyosin) of Dermatophagoides pteronyssinus, express and purify recombinant protein Der p 10, and analyze the immunogenicity and biological information of the protein. Methods Cultured mites were picked for total RNA extraction, then cDNA was generated by reverse transcription and underwent RT-PCR to amplify the Der p 10 gene. The pET-24a vector was ligated with the Der p 10 gene and transformed into the Top10 cloned bacteria. After BamH I and Xho I double enzyme digestion and sequencing, the construct was transformed into Escherichia coli BL21 (DE3), induced by 1 mmol/L IPTG, and examined in 12% SDS-PAGE to analyze the expression of recombinant protein. After purification by Ni⁺ ion affinity chromatography, the immunogenicity of Der p 10 recombinant protein was analyzed by Western blotting using serum of patient with dust mite allergy as the primary antibody. The ProtParam Tools, HNN, SWISS MODEL, DNAStar were used to predict the physiochemical properties of Der p 10, the secondary and tertiary structure of tropomyosin, and B cell epitope; Blastpand MEGA tool were used to construct Der p 10 phylogenetic tree. Results The target gene Der p 10 was obtained by RT-PCR, with an open reading frame of 855 bp, encoding 284 amino acids. The results of double enzyme digestion and sequencing showed that Der p 10 was linked to the vector. SDS-PAGE analysis showed that the relative molecular mass of Der p 10 recombinant protein was 38 000, which was a soluble protein. Western blotting analysis showed that the Der p 10 recombinant protein reacted with the serum of dust mite allergy patient. The bioinformatics analysis indicated that the Der p 10 protein was unstable, and its secondary and tertiary structures were mainly by α-helices. DNAStar predicted the presence of one B cell epitope peptide sequence in Der p 10. Blastp alignment and MEGA analysis showed that it was closely related to Der f 10, with sequence similarity reaching 94.4%. Conclusion The Der p 10 recombinant plasmid was constructed, and the expressed soluble recombinant protein of Der p 10 can specifically bind to the IgE antibody in patient serum with dust mite allergy, demonstrating its apparent immunogenicity.

Key words: Dermatophagois pteronyssinus, Der p 10, Expression, Purification, Immunological characteristics, Bioinformatics

CLC Number:

R384.4

GUO Chu-tong, OUYANG Chun-yan, HE Jun-xian, JI Shu-yu, YANG Li-teng, LIU Xiao-yu. Cloning, expression, purification and immunogenicity analysis of the tenth-class allergen of Dermatophagoides pteronyssinus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(6): 723-729.

Figures/Tables 5

References 22

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