Production of recombinant protein of Tyr p 32 from Tyrophagus putrescentiae and identifying its immunoreactivity
10.3760/cma.j.cn112150-20231225-00485
- VernacularTitle:腐食酪螨过敏原Tyr p 32重组蛋白的制备和免疫反应性鉴定
- Author:
Dongmei ZHOU
1
;
Yaning REN
;
Yuanfen LIAO
;
Ying ZHOU
;
Yubao CUI
Author Information
1. 南京医科大学附属无锡人民医院临床研究中心，无锡 214023
- Publication Type:Journal Article
- Keywords:
Tyrophagus putrescentiae;
Recombinant protein;
Tyr p 32;
IgE binding rate;
Immunoreactivity
- From:
Chinese Journal of Preventive Medicine
2024;58(12):1933-1939
- CountryChina
- Language:Chinese
-
Abstract:
The present study was aimed to produce the recombinant protein of Tyrophagus putrescentiae allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of T. putrescentiae 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 T.putrescentiae-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 T. putrescentiae-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 ( t=-29.10, P=0.001 2; t=-33.69, P=0.000 9), which also significantly increased the mRNA expression levels of IL-6 and IL-8 ( t=-9.15, P=0.011 7; t=-17.16, P=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.
