Guo Wei, Jiang Yu-Xin, Li Chao-Pin
Department of Medical Parasitology, Wannan Medical College, Wudu 241002, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Aug 30;30(4):274-8.
To express a chimeric gene R8 derived from the group 1 allergens of dust mites using prokaryotic expression system and detect their bioactivities.
PCR amplification was performed using specific primers of Derf1 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 BL21 and induced by 1 mmol/L isopropyl-beta-D-1-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 jl, 0.1 .tg/jl) and inhaled challenge as aerosol (0.5 microg/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 rDerf 1, rDerp 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 bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the level of interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) in BALF.
SDS-PAGE analysis revealed that chimeric gene R8 was expressed with a band of approximately M(r) 35 000. Compared with groups of rDerf 1 and rDer p 1 [(80.44 +/- 15.50) and (90.79 +/- 10.38) microg/ml, respectively], IgE binding capacity of the protein R8 (37.03 +/- 12.46) microg/ml was statistically lower (P < 0.001). The level of IFN-gamma 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 < or = 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).
Chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.
利用原核表达系统表达源自尘螨1类变应原的嵌合基因R8，并检测其生物活性。
以Derf1基因特异性引物和含R8嵌合基因的pUCm-T重组质粒为模板进行PCR扩增。PCR产物分别经限制性内切酶BamH I和Xho I双酶切后插入pET28a(+)空载体。重组质粒转入大肠杆菌BL21株，用1 mmol/L异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导。通过SDS-PAGE检测表达产物并纯化目标蛋白。用尘螨过敏患者血清通过ELISA检测纯化蛋白R8的IgE结合活性。为测定R8蛋白的免疫原性，将75只BALB/c小鼠随机分为5组，即PBS组（阴性对照组）、rDer f 1组和rDer p 1组（阳性组）、R8组和哮喘组。在第0、7、14天腹腔注射变应原（100 μl，0.1 μg/μl）对小鼠进行尘螨提取物处理，并在第21天进行7天的雾化吸入激发（0.5 μg/ml，30分钟/天）。在免疫治疗组第25 - 27天吸入前，分别用rDerf 1、rDerp 1和R8变应原进行特异性变应原免疫治疗。阴性对照组小鼠始终用PBS处理。最后一次激发24小时后，处死每组小鼠。收集支气管肺泡灌洗液（BALF）。用ELISA检测BALF中干扰素-γ（IFN-γ）和白细胞介素4（IL-4）水平。
SDS-PAGE分析显示嵌合基因R8表达出一条约M(r) 35 000的条带。与rDerf 1组和rDer p 1组[分别为(80.44 ± 15.50)和(90.79 ± 10.38) μg/ml]相比，蛋白R8的IgE结合能力（37.03 ± 12.46）μg/ml在统计学上较低（P < 0.001）。R8组血清中IFN-γ水平[(343.43 ± 38.79) pg/ml]高于PBS组和哮喘组[分别为(393.93 ± 50.68)和(208.44 ≤ 46.11)pg/ml]（P < 0.01），但与rDerf 1组和rDer p 1组相比无统计学差异（P > 0.05）。R8组IL-4水平明显低于其他组（P < 0.05或P < 0.01）。
源自尘螨1类变应原的嵌合蛋白R8已成功表达，具有低变应原性和高免疫原性。
