重组纳米级粉尘螨变应原第1组分的制备及其在哮喘小鼠中的作用研究

The preparation of recombinant Nanoscale Der f 1 allergen of Dermatophagoides farinae and its roles in mouse asthma

目的应用基因工程技术获得纳米级粉尘螨变应原第1组分s-layer/Der f 1,建立重组纳米级变应原的方法。方法利用S-layer/Strep-tag I这一具有自我组装能力的纳米模式母体嵌合粉尘螨变应原第1组分,并置大肠杆菌中表达,用分离纯化获得的表达产物免疫治疗哮喘小鼠,从分子、细胞、组织和器官4个层面证实该重组变应原诱导哮喘小鼠免疫耐受的可能性和有效性。结果采用基因工程技术将球形芽孢杆菌S层蛋白编码基因sllB的3′端去除600 bp后与尘螨主要变应原第1组分Der f 1连接,5′端与Strep-tag I的编码基因连接,成功构建克隆质粒pMD19-T-Strep-tag I/sllB1-2 706 bp/Der f 1,测序正确后,成功构建表达质粒pET28a(+)-Strep-tag I/sllB1-2 706 bp/Der f 1并转化E.coli BL21诱导表达,透射电镜显示该表达产物rDer f 1/S-layer位于工程菌表面,平均粒径为(116.43±14)nm。用获得的重组变应原rDer f 1、rDer f 1/S-layer免疫治疗哮喘小鼠,证实其可改善小鼠肺部炎症,降低支气管肺泡灌洗液中细胞总数、中性粒细胞、嗜酸性粒细胞和总IgE、螨特异性IgE水平,增加总IgG、螨特异性IgG水平,并上调外周血Th1型、下调Th2型细胞因子表达水平,并以重组纳米级变应原疗效更好,免疫调节作用更强。结论获得了纳米级粉尘螨变应原第1组份重组体,该制品可以诱导哮喘小鼠免疫耐受,具有较强的免疫原性,所建立的纳米级变应原原核表达体系为生产纳米药物和疫苗提供了一个新的途径。

The current study is aimed to develop a genetic method for recombinant allergen on nanometer levels.The gene sllB coding surface layer protein（S-layer） was truncated with 600 bp at the 3′ end,and then linked with the dust mite allergen Der f 1 at the 3′ end and 5′ with Strep-tag I to get a cloning plasmid pMD19-T-Strep-tag I/sllB1-2 706 bp/Der f 1.After being verified by sequencing,the expression plasmid pET28a（＋）-Strep-tag I/sllB1-2 706 bp/Der f 1 was transformed into E.coli BL21 and expressed with IPTG induction.By transmission electron microscopy,the expression product rDer f 1/S-layer was located on the surface of the engineering strain with avaerage diameter of（116.43±14） nm,which demonstrated that the expression system was developed successfully for recombinant nanometer-scale allergen.Then the recombinant allergen rDer f 1 and rDer f 1/S-layer was used to treat the asthmatic mouse model.The results from the experimental animals suggested that the two recombinant allergens can alleviate the lung inflammation,decrease the total cell numbers of bronchoalveolar lavage fluid,the numbers of neutrophils and eosinophils,and the levels of total IgE,the mite-specific IgE,the total IgG,mite-specific IgG in BALF,increase the levels of Th1 cytokines,and decrease the levels of Th2 cytokines.The therapeutic effect and immunoregulatory roles of the recombinant nano-level allergen rDer f 1/S-layer was better than that of the recombinant allergen rDer f 1.In brief,the recombinant product of the group 1 allergen of Dermatophagoides farinae is obtained on nanometer level,which can induce immune tolerance and have good immunogenicity.