Objective:To characterize the group-29 allergens from Dermatophagoides(D.)pteronyssinus and investigate their ability to cross-react with other group-29 allergens from D.pteronyssinus as well as those from D.farinae a...Objective:To characterize the group-29 allergens from Dermatophagoides(D.)pteronyssinus and investigate their ability to cross-react with other group-29 allergens from D.pteronyssinus as well as those from D.farinae and Tyrophagus putrescentiae.Methods:Der p 29,Der f 29,and Tyr p 29 cDNA sequences were amplified from total RNA isolated from D.pteronyssinus,D.farinae and Tyrophagus putrescentiae,respectively.Then they were cloned into the pET28a vector,expressed in Rosetta2(DE3)plysS,and purified using anion exchange chromatography.The IgE-binding rates of rDer p 29 were assessed by IgE Western blotting.The four epitopes of rDer p 29 were predicted,synthesized,and detected by IgE-ELISA.The cross-reactivity among the recombinant proteins rDer p 29,rDer f 29,and rTyr p 29 was investigated using dot blot and IgE-ELISA inhibition experiments.The allergens’physiochemical properties,amino acid sequences,and tertiary structures were also compared.Results:Der p 29 was successfully expressed in Rosetta2(DE3)plysS as a single,393-bp open reading frame.Western blotting showed that the purified rDer p 29 protein exhibited an IgE-binding rate of 100%when tested on patient sera.The following four Der p 29 epitopes were predicted and synthesized:37-45(EP1),57-69(EP2),75-80(EP3),and 104-117(EP4).IgE-ELISA tests on 20 D.pteronyssinus-positive sera yielded IgE-binding rates of 85%(rDer p 29),80%(EP1),55%(EP2),40%(EP3),and 55%(EP4),respectively.The dot blot experiments further confirmed cross-reactivity among the three group-29 proteins.When used as an inhibitor,rDer p 29 demonstrated an average cross-reactive inhibition rate of 49.7%against rDer f 29 and 54.4%against rTyr p 29.When rTyr p 29 was used as an inhibitor,it showed an average cross-reactive inhibition rate of 56.3%against rDer f 29.Conclusions:A recombinant protein,rDer p 29 with strong allergenicity was produced.Moreover,it was found that rDer p 29 cross-reacted with rDer f 29 and rTyr p 29,due to their highly homologous sequences and structures.These findings highlight the importance of considering inter-species epitope cross-reactivity when diagnosing and treating allergic diseases.展开更多
基金supported by Jiangsu Maternal and child health research project(no.F202068)the Taihu Lake talent plan(Top-Level,no.2020THRC-GD-7)The Project of Wuxi Health Commission(no.201949).
文摘Objective:To characterize the group-29 allergens from Dermatophagoides(D.)pteronyssinus and investigate their ability to cross-react with other group-29 allergens from D.pteronyssinus as well as those from D.farinae and Tyrophagus putrescentiae.Methods:Der p 29,Der f 29,and Tyr p 29 cDNA sequences were amplified from total RNA isolated from D.pteronyssinus,D.farinae and Tyrophagus putrescentiae,respectively.Then they were cloned into the pET28a vector,expressed in Rosetta2(DE3)plysS,and purified using anion exchange chromatography.The IgE-binding rates of rDer p 29 were assessed by IgE Western blotting.The four epitopes of rDer p 29 were predicted,synthesized,and detected by IgE-ELISA.The cross-reactivity among the recombinant proteins rDer p 29,rDer f 29,and rTyr p 29 was investigated using dot blot and IgE-ELISA inhibition experiments.The allergens’physiochemical properties,amino acid sequences,and tertiary structures were also compared.Results:Der p 29 was successfully expressed in Rosetta2(DE3)plysS as a single,393-bp open reading frame.Western blotting showed that the purified rDer p 29 protein exhibited an IgE-binding rate of 100%when tested on patient sera.The following four Der p 29 epitopes were predicted and synthesized:37-45(EP1),57-69(EP2),75-80(EP3),and 104-117(EP4).IgE-ELISA tests on 20 D.pteronyssinus-positive sera yielded IgE-binding rates of 85%(rDer p 29),80%(EP1),55%(EP2),40%(EP3),and 55%(EP4),respectively.The dot blot experiments further confirmed cross-reactivity among the three group-29 proteins.When used as an inhibitor,rDer p 29 demonstrated an average cross-reactive inhibition rate of 49.7%against rDer f 29 and 54.4%against rTyr p 29.When rTyr p 29 was used as an inhibitor,it showed an average cross-reactive inhibition rate of 56.3%against rDer f 29.Conclusions:A recombinant protein,rDer p 29 with strong allergenicity was produced.Moreover,it was found that rDer p 29 cross-reacted with rDer f 29 and rTyr p 29,due to their highly homologous sequences and structures.These findings highlight the importance of considering inter-species epitope cross-reactivity when diagnosing and treating allergic diseases.
