Recombinant Expression And Identification Of Ovary Development-related Protein EJO1(Eris 2),an Allergen In The Ovary Of Eriocheir Sinensis

Objective Crab is a major shellfish allergen. Most studies have focused on allergens in crab muscle(CM) rather than on allergens in crab ovary(CO). This study took CO as research objects, aimed to identify potential allergens in CO from Eriocheir sinensis(E.sinensis), and evaluated the possibility of CO as a potential source of crab allergens. One of the newly discovered allergens has been recombinant expressed, evaluated its immunological activity and biological activity, and discussed whether the recombinant allergen can be used as a candidate allergen for the diagnosis and treatment of crab allergy.Methods Analysis of allergic-components from the ovary of Eriocheir sinensis: CO and CM were each ground up and the cells were lysed in lysis buffer. The components of CO and CM were analyzed by SDS-PAGE. The differential reactivity of CM and CO extracts to positive sera from patients allergic to crabs were analyzed by Dot blotting.The allergic protein were analyzed by Immunoblotting and 2DE immunoblotting, and identified by MS/ MS. These aimed to confirmed the possibility of CO as a potential source of crab allergens.Recombinant expression and identification of ovary development-related protein EJO1(Eris 2): First of all, we lysed the total RNA of the ovary of E.sinensis by Trizol lysis, gained the gene of Eris 2 by amplification, and connected the gene and the p ET-28 b(+) plasmid to construct a recombinant plasmid. Second, recombinant Eris 2(r Eris 2) were expressed by Rosetta(DE3) Competent Cell and subsequently purified using a Ni-NTA column. Third, the reactivity of r Eris 2 to positive sera were checked by by ELISA, inhibition ELISA, immunoblot inhibition experiment and Basophil activation test. These aimed to confirmed r Eri 2 s as a kind of recombinant proteins used in E.sinensis allergy diagnosis.Results Analysis of allergic-components from the ovary of Eriocheir sinensis:SDS-PAGE confirmed the integrality of the protein components of CO and CM. Dot blotting results revealed the differential reactivity of CM and CO extracts to positive sera from patients allergic to crabs. Three CO proteins showed good specific Ig E-binding activities at molecular weight(MW) 105 k Da and p I 7.5, MW 85 k Da and p I 8, MW 70 k Da and p I 5.5, and MW 28 k Da and p I 6, MW 28 k Da and p I 6.2in2DE immunoblotting analysis. Mass spectrometry identified the proteins as vitellogenin, hemocyanin, ovary development-related protein EJO1 and ovary development-related protein EJO2.Recombinant expression and identification of Eris 2: After expression and purification, the recombinant allergen Eris 2 showed a single major band migrating at about 32 k Da. r Eris 2 exhibited obviously higher reactivity to 29 positive sera than to 6negative sera by ELISA. Pre-incubation with r Eris 2 abrogated the reactivity of positive sera to CO by inhibition ELISA and immunoblot inhibition experiment. Moreover, co-incubation of recombinant Eris 2 with patient basophils dose-dependently promoted basophil activation, confirming the biological activity of Eris 2.Conclusion The results confirmed that crab ovary tissue contains a variety of allergen components, including at least vitellogenin, hemocyanin and ovary development-related protein EJO1(Eris 2) and ovary development-related protein EJO2. Recombinant Eris 2 protein has good reactivity with serum s Ig E, and it can be used as an antigen in serum s Ig E test. Recombinant Eris 2 also can be used for basophils activation test as a important activator, and it could evaluate he degree of risk when the body contact with the allergen or the food.