[Production of recombinant protein of Tyr p 32 from <i>Tyrophagus putrescentiae</i> and identifying its immunoreactivity].

The present study was aimed to produce the recombinant protein of <i>Tyrophagus putrescentiae</i> allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of <i>T. putrescentiae</i> and inserted into pET-28a (+) vector. The constructed plasmid pET-28a (+)-Tyr p 32 was transformed into BL21 (DE3) receptor cells. After being induced with IPTG, the recombinant protein was purified with Ni column, and then identified on SDS-PAGE and Western blotting. Serum of children with allergic asthma and(or) rhinitis was collected, IgE-ELISA and IgE-Western blotting were used to detect the binding rate of rTyr p 32 to human <i>T.putrescentiae-</i>positive serum. After human bronchial epithelial cells BEAS-2B being cultured with rTyr p 32, the expression levels of IL-6 and IL-8 cytokines was detected by ELISA and qRT-PCR, t-test was used for pairwise comparison between groups. The results showed that the cDNA length of Tyr p 32 was 885 bp. The sequence identity between Tyr p 32 and Der p 32, Der f 32 was 70.21% and 68.03%, respectively. According to SDS-PAGE and Western blotting, the molecular weight of rTyr p 32 was about 35 000 Da, which was consistent with the theoretical value. IgE-ELISA results showed that the positive rate of rTyr p 32 was 41.38% (12/29) against <i>T. putrescentiae</i>-positive serum. When BEAS-2B cells were cultured with rTyr p 32, the expression of IL-6 and IL-8 increased in the cell supernatant in a dose-dependent manner (<i>t</i>=-29.10,<i>P</i>=0.001 2;<i>t</i>=-33.69,<i>P</i>=0.000 9), which also significantly increased the mRNA expression levels of IL-6 and IL-8 (<i>t</i>=-9.15,<i>P</i>=0.011 7;<i>t</i>=-17.16,<i>P</i>=0.003 4). In conclusion, the recombinant protein rTyr p 32 was successfully prepared, which provides raw materials for component diagnosis and specific immunotherapy of allergic diseases.