AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori(H pylori)isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies i...AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori(H pylori)isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori.METHODS: H pylori strains in biopsy specimens from 157patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected.The target recombinant proteins rUreB, rVacA, rCagA1,rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography.Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA,rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody,expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed.RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2%respectively. In the 125 serum samples from the H pyloriinfected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were 100%, 42.4%, 89.6%, 81.6%, 93.6%, 98.4%and 92.8% respectively. H pylori strains were isolated from 79.6% (125/157) of the biopsy specimens, but no close correlations among the H pylori infection frequencies and different types of chronic gastritis and peptic ulcer could be found (P＞0.05, x2 = 0.01-0.87). The VacA positive rate (82.40%) in the strains isolated from the specimens of patients with peptic ulcer and the anti-VacA positive rate (54.3%) in the sera from the patients were significantly higher than those (51.5%, 32.3%) from the patients with chronic gastritis (P＜0.01, x2 = 13.19; P＜0.05, x2 = 6.13).When analysis was performed in the different types of chronic gastritis, the VacA in the strains isolated from the specimems of patients with active gastritis showed a higher expression frequency (90.0%) than those from superficial (47.9%) and atrophic gastritis (30.0%) (P＜0.05, x2 = 5.93;P＜0.01,x2 = 7.50). While analysis was carried out in the strains isolated from the specimens with superficial (93.8%) and active gastritis (100%), NapA showed a higher expression frequency compared to that from atrophic gastritis (60.0%) (P＜0.01, x2 = 8.88; P＜0.05,x2= 5.00).CONCLUSION: The types of chronic gastritis and peptic ulcer and their severity are not associated with H pylori infection frequency but closely related to the infection frequency of different virulent H pylori strains. The optimal antigens for developing vaccine and diagnostic kit are UreB,FlaA, HpaA, FlaB, NapA and CagA1, but not VacA.展开更多
为探究湿热处理对花生球蛋白Ara h 3抗原性的影响,采用硫酸铵分级沉淀法从花生脱脂粉中分离纯化出Ara h 3,进行不同温度、时间和质量浓度的湿热处理,通过正交试验确定最优湿热处理条件,采用间接ELISA法检测湿热处理后Ara h 3抗原性变化...为探究湿热处理对花生球蛋白Ara h 3抗原性的影响,采用硫酸铵分级沉淀法从花生脱脂粉中分离纯化出Ara h 3,进行不同温度、时间和质量浓度的湿热处理,通过正交试验确定最优湿热处理条件,采用间接ELISA法检测湿热处理后Ara h 3抗原性变化,测定外源荧光、游离巯基含量以及圆二色光谱,从蛋白结构的变化解释湿热处理对Ara h 3抗原性的影响。研究表明:湿热处理能够降低Ara h 3抗原性,且在质量浓度10 mg/mL、90℃处理30 min时,抗原性降至最低;湿热处理后的Ara h 3荧光强度、表面疏水性增强,三级结构发生变化;游离巯基含量增加、空间结构发生改变;α-螺旋和β-折叠含量降低,无规则卷曲含量增加,二级结构发生变化。湿热处理后Ara h 3空间结构发生改变,部分抗原表位遭到破坏或被包埋,抗原性降低。展开更多
为探讨辐照处理对花生Ara h 2蛋白结构与致敏活性的影响,采用不同剂量^(60)Co-γ辐照处理分离纯化所得到的花生过敏原Ara h 2蛋白,结合紫外扫描光谱、圆二色谱(CD)和聚丙烯酰胺凝胶电泳(SDS-PAGE)评估辐照处理后Ara h 2蛋白的结构变化,...为探讨辐照处理对花生Ara h 2蛋白结构与致敏活性的影响,采用不同剂量^(60)Co-γ辐照处理分离纯化所得到的花生过敏原Ara h 2蛋白,结合紫外扫描光谱、圆二色谱(CD)和聚丙烯酰胺凝胶电泳(SDS-PAGE)评估辐照处理后Ara h 2蛋白的结构变化,并用免疫印迹法和间接酶联免疫吸附法检测辐照处理后Ara h 2的抗原性变化。结果表明,^(60)Co-γ辐照处理可以显著改变花生Ara h 2蛋白的构象,使其降解、发生交联。随着辐照剂量的增大,Ara h 2蛋白与抗体的结合能力呈逐渐下降趋势,且与蛋白紫外吸光度的增强和α-螺旋含量的降低呈现良好的相关性。当辐照剂量为10 kGy时,可基本破坏Ara h 2蛋白的结构和免疫活性。^(60)Co-γ辐照处理可以有效降低花生过敏原Ara h 2蛋白的致敏性,这为花生脱敏技术的研究提供了新思路。展开更多
从花生种子中分离纯化花生过敏原Ara h 2,采用酶联免疫吸附试验(ELISA)、圆二色谱(CD)、ANS荧光探针及紫外可见(UV-Vis)光谱等方法,系统研究热加工对Ara h 2抗原性和结构的影响。结果表明:Ara h 2蛋白经55或70℃处理后其抗原性略有升高,...从花生种子中分离纯化花生过敏原Ara h 2,采用酶联免疫吸附试验(ELISA)、圆二色谱(CD)、ANS荧光探针及紫外可见(UV-Vis)光谱等方法,系统研究热加工对Ara h 2抗原性和结构的影响。结果表明:Ara h 2蛋白经55或70℃处理后其抗原性略有升高,经85,100或115℃处理后,其抗原性显著降低,且随着温度和时间的增加其抗原性均不断降低。CD色谱分析表明,Ara h 2经热处理后其二级结构发生变化;ANS荧光探针光谱显示,不同的热处理均导致Ara h 2表面疏水性增加。紫外光谱显示,不同温度对Ara h 2处理30 min后(除55℃外),其紫外吸收值均升高。Ara h 2经100℃处理不同时间后,其紫外吸收值均有增加。由此推断,花生过敏原Ara h 2的构象改变导致了其抗原性的降低。展开更多
基金Supported by the Excellent Young Teacher Fund of Chinese Education Ministry and the General Science Technology Research Program of Zhejiang Province, No. 001110438
文摘AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori(H pylori)isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori.METHODS: H pylori strains in biopsy specimens from 157patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected.The target recombinant proteins rUreB, rVacA, rCagA1,rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography.Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA,rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody,expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed.RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2%respectively. In the 125 serum samples from the H pyloriinfected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were 100%, 42.4%, 89.6%, 81.6%, 93.6%, 98.4%and 92.8% respectively. H pylori strains were isolated from 79.6% (125/157) of the biopsy specimens, but no close correlations among the H pylori infection frequencies and different types of chronic gastritis and peptic ulcer could be found (P＞0.05, x2 = 0.01-0.87). The VacA positive rate (82.40%) in the strains isolated from the specimens of patients with peptic ulcer and the anti-VacA positive rate (54.3%) in the sera from the patients were significantly higher than those (51.5%, 32.3%) from the patients with chronic gastritis (P＜0.01, x2 = 13.19; P＜0.05, x2 = 6.13).When analysis was performed in the different types of chronic gastritis, the VacA in the strains isolated from the specimems of patients with active gastritis showed a higher expression frequency (90.0%) than those from superficial (47.9%) and atrophic gastritis (30.0%) (P＜0.05, x2 = 5.93;P＜0.01,x2 = 7.50). While analysis was carried out in the strains isolated from the specimens with superficial (93.8%) and active gastritis (100%), NapA showed a higher expression frequency compared to that from atrophic gastritis (60.0%) (P＜0.01, x2 = 8.88; P＜0.05,x2= 5.00).CONCLUSION: The types of chronic gastritis and peptic ulcer and their severity are not associated with H pylori infection frequency but closely related to the infection frequency of different virulent H pylori strains. The optimal antigens for developing vaccine and diagnostic kit are UreB,FlaA, HpaA, FlaB, NapA and CagA1, but not VacA.
文摘为探究湿热处理对花生球蛋白Ara h 3抗原性的影响,采用硫酸铵分级沉淀法从花生脱脂粉中分离纯化出Ara h 3,进行不同温度、时间和质量浓度的湿热处理,通过正交试验确定最优湿热处理条件,采用间接ELISA法检测湿热处理后Ara h 3抗原性变化,测定外源荧光、游离巯基含量以及圆二色光谱,从蛋白结构的变化解释湿热处理对Ara h 3抗原性的影响。研究表明:湿热处理能够降低Ara h 3抗原性,且在质量浓度10 mg/mL、90℃处理30 min时,抗原性降至最低;湿热处理后的Ara h 3荧光强度、表面疏水性增强,三级结构发生变化;游离巯基含量增加、空间结构发生改变;α-螺旋和β-折叠含量降低,无规则卷曲含量增加,二级结构发生变化。湿热处理后Ara h 3空间结构发生改变,部分抗原表位遭到破坏或被包埋,抗原性降低。
文摘为探讨辐照处理对花生Ara h 2蛋白结构与致敏活性的影响,采用不同剂量^(60)Co-γ辐照处理分离纯化所得到的花生过敏原Ara h 2蛋白,结合紫外扫描光谱、圆二色谱(CD)和聚丙烯酰胺凝胶电泳(SDS-PAGE)评估辐照处理后Ara h 2蛋白的结构变化,并用免疫印迹法和间接酶联免疫吸附法检测辐照处理后Ara h 2的抗原性变化。结果表明,^(60)Co-γ辐照处理可以显著改变花生Ara h 2蛋白的构象,使其降解、发生交联。随着辐照剂量的增大,Ara h 2蛋白与抗体的结合能力呈逐渐下降趋势,且与蛋白紫外吸光度的增强和α-螺旋含量的降低呈现良好的相关性。当辐照剂量为10 kGy时,可基本破坏Ara h 2蛋白的结构和免疫活性。^(60)Co-γ辐照处理可以有效降低花生过敏原Ara h 2蛋白的致敏性,这为花生脱敏技术的研究提供了新思路。
文摘从花生种子中分离纯化花生过敏原Ara h 2,采用酶联免疫吸附试验(ELISA)、圆二色谱(CD)、ANS荧光探针及紫外可见(UV-Vis)光谱等方法,系统研究热加工对Ara h 2抗原性和结构的影响。结果表明:Ara h 2蛋白经55或70℃处理后其抗原性略有升高,经85,100或115℃处理后,其抗原性显著降低,且随着温度和时间的增加其抗原性均不断降低。CD色谱分析表明,Ara h 2经热处理后其二级结构发生变化;ANS荧光探针光谱显示,不同的热处理均导致Ara h 2表面疏水性增加。紫外光谱显示,不同温度对Ara h 2处理30 min后(除55℃外),其紫外吸收值均升高。Ara h 2经100℃处理不同时间后,其紫外吸收值均有增加。由此推断,花生过敏原Ara h 2的构象改变导致了其抗原性的降低。
