Identification Of Artemisia Species And Analysis Of Allergens Of Artemisia Lavandulaefolia In North China

Objective: Pollinosis is a common type 1 allergic reaction mainly involving the respiratory tract and the eye.It is caused by pollen in wind-pollinated and has obvious seasonal and regional characteristics.Its incidence increases year by year,seriously affecting people's health and increasing social and economic burden.The pollinosis caused by Artemisia pollen in northern China is particularly prevalent.Therefore,based on the existing research on Artemisia pollen,the identification of Artemisia species is carried out,and the immune activity of Artemisia allergens is further analyzed.It is of great value for the diagnosis and treatment of pollinosis in northern China.Methods: In this study,through pollinosis questionnaire,allergen screening,field pollen and serum collection,the existing nucleic acid sequence database was used to identify plant species by ITS sequence analysis and morphology.The Artemisia lavandulaefolia pollen allergen genes Art la 1 to Art la 6 were synthesized by gene synthesis techniques.The N.benthamiana plant,P.pastoris and E.coli protein expression vector were constructed,transformed into a host for recombinant expression of the allergen,and purified by metal nickel ion ligand affinity chromatography.Finally,SDS-PAGE electrophoresis analysis,and western blotting test of the serum of Artemisia and c-myc label were used to verify the lgE binding activity of recombinant allergen.Results:(1)The prevalence of pollinosis in the northern Chinese aviation unit is 32.78%(376/1147),which is a high incidence in summer and autumn.The results of SPT and serum slgE showed that Artemisia was one of the main sources of sensitization in the region,and the positive detection rate of aviation units was as high as 33.24% and 48.84%;.(2)The ITS sequences and morphological identifications were Artemisia lavandulifolia(KX421740),Artemisia sieversiana(KX421708),Artemisia annua(KX421777),Artemisia argyi(JX051681)and Artemisia scoparia(KX421761).(3)N.benthamiana successfully expressed and purified allergens Art la 1 and Art la 2,,and the leaves showed green fluorescence by fluorescence microscopy.(4)Allergens Art la 3 and Art la 6 were recombinantly expressed by P.pastoris induced methanol and purified,SDSPAGE analysis showed that Art la 3 has a small amount of low-purity protein,Art la 6 has no protein.(5)E.coli recombinant expression and purification of allergen Art la 4 and Art la 5 are highly expressed by SDS-PAGE;(6)Western Blotting assay confirms the expression of recombinant protein,the results showed that allergens Art la 1,Art la 2,Art la 3,and Art la 4 were positive for c-myc tag antibody binding,negative for allergen Art la 5 and Art la 6;(7)The results of Western Blotting of the serum of 60 patients positive for Artemisia showed that the positive rates of recombinant allergen Art la 1,Art la 2,Art la 3 and Art la 4 bound to serum lgE were 55.0%,31.7%,30.0% and 33.4%,respectively.Conclusion: The prevalence of pollinosis in northern China is as high as 39.15%,mainly in summer and autumn.Artemisia lavandulaefolia is one of the main sources of sensitization.In the N.benthamiana,P.pastoris and E.coli,four major allergens of Artemisia lavandulaefolia pollen were successfully recombined and purified,namely Art la 1,Art la 2,Art la 3.0102 and Art la 4,and with serum IgE has immunological binding activity,which lays a foundation for the preparation of diagnostic reagents and allergen immunotherapy.