Abstract: Objective To establish a prokaryotic expression system and confirm the temperature stress response function of HSP16-1 of Dermatophagoides farinaeat protein level. Methods Based on the HSP16-1 gene sequence of D. farinae obtained by previous RNA-seq,specific primers were designed,amplified by PCR,cloned and sequenced. Bioinformatics software was used to analyze the physical and chemical properties,subcellular location and three-dimensional structure. The prokaryotic expression of plasmid pET32a / HSP16-1 was constructed and transformed into E. coli BL21. The expression of HSP16-1 recombinant protein was induced by 1 mmol / L IPTG at 16 ℃ ,28 ℃ and 37 ℃ ,and the samples were collected for SDS-PAGE analysis at 2 h, 4 h, 6 h and 8 h, respectively. The bacteria growth curves of the recombinant strains (pET32a / HSP16-1) and control strains (pET32a) under heat and cold stress were drawn. Results The sequencing results showed that the complete coding sequence ( CDS) of D. farinae HSP16-1 was 462 bp, which encoded 153 amino acids. BLAST alignment demonstrated that the nucleotide and amino acid sequences of D. farinae HSP16-1 were in similarity by 84. 63% and 87. 58%,respectively,with the closely related species D. pteronyssinus . Subcell were localized in the nucleus,and conservative region prediction revealed conservative region of α crystallin HSP23 superfamily. The recombinant plasmid pET32a / HSP16-1 was successfully constructed and identified by bacteria liquid PCR and double restriction enzyme digestion. SDS-PAGE analysis showed that HSP16-1 protein was successfully expressed,and the best induction condition was 37 ℃ for 6 h. The bacterial growth curve indicated that the growth of pET32a / HSP16-1 recombinant strains was better than that of pET32a control strains under heat stress,yet the growth of control strains was better than that of recombinant strains under cold stress. Conclusion HSP16-1 protein of D. farinae only generate stress response function under heat stress,yet does not show similar function under cold stress.

Key words: Dermatophagoides farinae, HSP16-1, Prokaryotic expression, Temperature stress response, Functional identification