屋尘螨第13组变应原克隆表达、纯化及免疫原性鉴定

目的 克隆表达及纯化屋尘螨(Dermatophagoides pteronyssinus,Der p)第13组变应原(Der p13)蛋白,并鉴定其免疫原性.方法 提取屋尘螨总RNA,通过逆转录聚合酶链式反应(RT-PCR)技术获得Der p13的cDNA片段.正确设计引物,利用已获得的cDNA片段进行PCR扩增,获得...

Cloning,expression,purification and immunogenicity identification of the recombinant Allergen Der p13 from Dermatophagoides pteronyssinus

Objective To clone,express and purify the 13 th group of allergen(Der p13)protein from Dermatophagoides pteronyssinus,and to identify its immunogenicity.Methods The total RNA of Dermatophagoides pteronyssinus was extractedand cDNA fragment of Der p13 was obtained by RT-PCR.The primers were designed correctly,and Der p13 gene was obtainedby PCR amplification using the obtained cDNA fragment.The amplification products were linked to the vector and transferredinto E.coli TOP10.The positive colonies were picked out and retained.The simples of the positive colonies were sent forsequencing.The recombinant protein Der p13 was purified by Ni column affinity chromatography and induced by IPTG.Thepurified products were identified by 12 alkyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.Results pET-24 a-Der p13 was successfully constructed.SDS-PAGE showed that the expression of soluble protein in thesupernatant was high and the molecular weight was about 13000.The results of Western blotting showed that the protein couldbind to the sera of patients with dust mite allergy and had certain immunogenicity.According to the data about antigenic index,hydrophilicity plot,surface probability and flexible regions of DNA Star,the antigenic of Der p13 could be deduced.Thephylogenetic tree analysis of Der p13 showed that Dermatophagoides pteronyssinus had high homology with Dermatophagoides farina,Sarcoptes scabiei,Lepidoglyphus destructor and Acarus siro.Conclusion The recombinant protein of Der p13 wassuccessfully cloned,expressed and purified.The protein has some immunogenicity,which lays a foundation for clinicalexperimental study of the diagnosis and treatment of allergic diseases.