Construction Of Humanized Mouse IL-17A&IL-17F

Psoriasis is a common autoimmune deficiency chronic inflammatory skin disease in men and women around the world,and interleukin-17A(IL-17A)and interleukin-17F(IL-17F)play a role in inflammatory diseases such as psoriasis.important role.Therefore,targeted cytokine therapy for human IL17A and IL-17F has become one of the important strategies for the treatment of psoriasis.The purpose of this study is to construct IL-17A/IL-17F double humanized mice(hIL-17A/hIL-17F)and verify the relevant phenotypes of humanized mice.By consulting relevant literature and data,determining the genetic information of IL-17A and IL-17F,designing a humanized mouse targeting strategy,so as to construct,prepare and screen efficient and specific sgRNA,and then design an effective Southern blot strategy to construct,Prepare and screen out the plasmids that meet the targeting requirements,and finally microinject the successfully constructed plasmids into the mouse fertilized eggs.After breeding,perform primer ligation verification experiments on the primary chimeric mice,eliminate negative mice,screen positive mice,and pass Selfcrossing and backcrossing methods to expand the population.After that,the amplified mice were subjected to primer ligation verification experiment and Southern blotting again to confirm that there was no random insertion,and the positive heterozygous mice meeting the requirements were screened out.The heterozygous mice were collected,and their phenotypes were analyzed by ELISA to further confirm the humanized protein expression of the heterozygous mice.The human IL-17A protein concentration in serum was 799.476 pg/mL,and the human IL-17F protein concentration in cell culture supernatant was 1598.92 pg/mL.After successful verification of heterozygous mice,the population will be expanded,and IL-17A/IL-17F double-derived homozygous mice that can be stably inherited are constructed.Afterwards,RTPCR and ELISA were used to further confirm whether the humanized protein of homozygous mice was transcribed and translated correctly.The protein concentration of human IL-17A in serum was 331.48 pg/mL.In this regard,successfully constructed hIL-17A/hIL-17F mice that can be inherited stably,providing a phenotypic-verified mouse humanized psoriasis model that can be used for research and development against psoriasis.This new type of therapeutic drug for specific cytokines can induce the preparation of a variety of human disease models for testing the in vivo efficacy of human specific antibodies.