两种尘螨1类变应原嵌合基因的原核表达及生物活性鉴定

Prokaryotic Expression of Chimeric Gene Derived from the Group 1 Allergens of Dust Mites and Bioactivity Identification

目的原核表达尘螨1类变应原（粉尘螨1类变应原仇叩和户尘螨1类变应原Derpl基因）的嵌合基因R8，并检测其生物活性。方法以含嵌合基因R8的pUCm-T重组质粒为模板，用Derel的特异性引物进行PCR扩增，产物经酶切后与载体pET28a（＋）连接，连接产物转入大肠埃希菌（E.coli）BL21感受态细胞，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达后纯化，十二烷基磺酸钠．聚丙烯酰胺凝胶电泳（SDS．PAGE）检测；纯化后的蛋白用ELISA检测其与粉尘螨过敏患者血清（IgE）的结合能力，同时以重组rDerfl和rDerP1蛋白作为对照。为检验嵌合蛋白R8的免疫原性，将75只BALB／c小鼠随机均分为5组，分别为PBS组（阴性对照组）、rDerf1组、rDerP1组、R8组和哮喘组（阳性对照组）。除PBS组外，其余各组分别于第0、7和14天每鼠腹腔注射（0．1μg/μl）粉尘螨注射液100μ1，第21～27天每鼠每天雾化吸入0．5μg／ml粉尘螨注射液悬浊液，30min／d。rDerf1组、rDerP1组和R8组于第25～27天雾化前30min．分别腹腔注射100μg／ml的rDerf1、rDerP1和R8蛋白各200μl进行特异性免疫治疗，PBS组用等量PBS进行腹腔注射和雾化吸人。所有小鼠于第27天雾化吸入后24h气管切开，用1mlPBS进行肺泡灌洗，收集肺泡灌洗液（BALF），检测γ干扰素（IFN-γ）和白细胞介素4（IL-4）的水平。结果SDS-PAGE结果显示，R8表达产物的蛋白相对分子质量（Mr）约为35000。EUSA检测结果显示，嵌合蛋白R8与粉尘螨过敏患者血清IgE的结合力为（37．03±12．46）μg／ml，显著低于rDerf1[（80．44±15．50）μg／ml]和rDerP1[（90．79±10．38）μg／ml]（P〈0．01）。动物实验结果显示，R8组IFN-γ水平[（343．43±38．79）pg／ml]与rDerfl组[（322．98±30．36）pg／m1]和rDerP1组[（314．97±33．89）pg/ml]的相比，差异均无统计学意义（P〉0．05）；与PBS组[（393．93±50．68）pg/ml]和哮喘组[（208．44±46．11）pg／m1]相比，差异均有统计学意义（P〈0．01）。R8组IL4水平显著低于其他各组水平（P〈0．05或0．01）。结论表达了具有低变应原性和高免疫原性的尘螨1类变应原嵌合基因R8。

Objective To express a chimeric gene R8 derived from the group 1 allergens of dust mites using prokaryotic expression system and detect their bioactivities. Methods PCR amplification was performed using specific primers of Derfl gene and the pUCm-T recombinant plasmid containing the R8 chimeric gene as a template. The PCR products were inserted into the pET28a（＋） empty vector after double digestion using restriction endonuclease BamH Ⅰ and Xho Ⅰ, respectively. The recombinant plasmid was transferred into E. coli line BI21 and induced by 1 mmol/L isopropyl-β- D-l-thiogalactopyranoside （IPTG）. The expressed product was detected by SDS-PAGE and the target protein was purified. IgE binding assay of the purified protein R8 was detected by ELISA using dust mite allergic patient sera. For determining immunogenicity of R8 protein, 75 BALB/c mice were randomly divided into 5 groups, namely PBS （negative control）, rDer f 1 group and rDer p 1 group （positive groups）, R8 group and asthma group. The mice were treated with dust mite extract at 0, 7, 14 day by intraperitoneal injection of allergens （100 μ1, 0.1 μg/μl） and inhaled challenge as aerosol（0.5 μg/ml, 30 min/d） on day 21 for 7 days. Before inhalation in immunotherapy groups at 25--27 day, specific allergen immunotherapy was performed using rDer f 1, rDer p 1 and R8 allergens respectively. Mice in negative control group were treated with PBS all the time. Twenty-four hours after the last challenge, mice in every group were sacrificed. The bron- choalveolar lavage fluid （BALF） was collected. ELISA was used to detect the level of interferon-γ（IFN-γ） and interleukin 4 （IL-4） in BAEF. Results SDS-PAGE analysis revealed that chimeric gene R8 was expressed with a band of approximately Mr 35 000. Compared with groups of rDerf 1 and rDer p 1 [（80.44±15.50） and （90.79±10.38）μg/ml, respectively], IgE binding capacity of the protein R8 （37.03±12.46）μg/ml was statistically lower （P〈0.001）. The level of IFN-γ in sera of R8 group [（343.43±38.79）pg/ml] was higher than that of the PBS and asthma groups [（393.93±50.68） and （208.44±46.11）pg/ml, respectively] （P〈0.01）, but no statistical difference to that of the rDerf 1 and rDer p 1 groups （P〉0.05）. IL-4 level in R8 group was lower markedly than the others （P〈0.05 or P〈0.01）. Conclusion Chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.