Identification and characterization of the Cul t 1 as major allergen from biting midge Culicoides tainanus

Midges are widely distributed globally. They can transmit numerous serious diseases as well as trigger an allergic reaction in the host. Their saliva contains a variety of proteins that act as sensitizers to stimulate the host's immune response, leading to IgE-mediated allergic symptoms.

In the present study, we constructed a mouse sensitization model with the thorax extract of the Culicoides tainanus, and evaluated the sensitization model by behavior and specific antibody expression. SDS-PAGE/western blot was used to detect the binding proteins by IgE antibody in the thorax extract of the C. tainanus. The objective band was cut for mass spectrometry to preliminarily clarify the potential allergen. The pET21a-Cul t 1 recombinant expression vector was constructed and the target protein was purified by Ni affinity chromatography. The sensitizing effect of the sensitizer was verified in vitro and in vivo.

Immunoblot analysis revealed that a 66 kDa protein (Cul t 1) from the chest extracts of C. tainanus could bind to serum IgE of sensitized mice. Cul t 1 was further identified by fragmentation, mass spectrometry and bioinformatics as maltase, an enzyme involved in sugar digestion. In vivo validation of the Cul t 1-mouse sensitization model showed that the scratching behavior of the Cul t 1 sensitized group was significantly higher than that of the control group according to behavioral evaluation. The results of HE staining of the skin of the injected area showed that Cul t 1 sensitized group was found to have a large number of inflammatory cell infiltration and increased fibrosis in the dermis. The ELISA detection of IgE showed that the Cul t 1 sensitized group was significantly elevated from the beginning of the 28th day onwards, and the expression level of IgE had a tendency to slow down with the prolongation of the sensitization time. The ELISA assay showed that the expression level of IgG1 increased significantly in the Cul t 1 group on day 42, while the results of the specific antibody IgG2a showed that there was no significant difference in both the Cul t 1 sensitized group and the control group. The results suggest that the immune response induced by Cul t 1 sensitized mice may be shifted toward a Th2 type immune response.

In this study, we identified the sensitizer of the C. tainanus, named Cul t 1. We successfully constructed the recombinant expression vector pET21a-Cul t 1, purified the Cul t 1 sensitizer by affinity chromatography, and verified the sensitizing effect of Cul t 1 in mice. The results laid a practicable foundation for the subsequent immune therapy of midge bites and bite control.