目的预测疥螨猪变种转录组中过敏原蛋白,对Sar s 14.3过敏原蛋白进行生物特性分析、原核表达并建立间接ELISA诊断方法。方法利用转录组测序(RNA-Seq)及BLAST、ProtParam、I-TASSER和COMPARE等软件和数据库对疥螨猪变种转录组中的过敏原...目的预测疥螨猪变种转录组中过敏原蛋白,对Sar s 14.3过敏原蛋白进行生物特性分析、原核表达并建立间接ELISA诊断方法。方法利用转录组测序(RNA-Seq)及BLAST、ProtParam、I-TASSER和COMPARE等软件和数据库对疥螨猪变种转录组中的过敏原组分进行预测并对疥螨Sar s 14.3过敏原蛋白序列进行理化性质分析、结构分析、同源性分析和间接ELISA诊断方法的建立。结果在疥螨猪变种的18980个转录本中共预测出390条过敏原序列,且随机匹配可能性值(E-value)为0的序列有28条。Sar s 14.3过敏原蛋白的理论相对分子质量为38 kDa,蛋白较稳定,为亲水性蛋白;二级结构以无规卷曲和β-折叠为主,其氨基酸序列与疥螨其他变种主要过敏原组分14蛋白序列相似性不低于99.76%,而与尘螨属螨虫的相似性最高为73.08%,与现行的形态学分类系统一致。所建立的间接ELISA诊断方法的最佳包被抗原浓度为2μg/mL,血清最佳稀释倍数是1∶400。结论本试验成功预测了疥螨猪变种转录组中的过敏原序列并克隆、表达了疥螨猪变种Sar s 14.3蛋白,初步预测了其分子特征,并以该过敏原蛋白为基础成功建立了一种快速的间接ELISA检测方法,可用于疥螨病的初步诊断和流行病学调查。展开更多
Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary es...Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary established the simple enzyme-linked immunosorbent assay (ELISA) without deconjugation, then developed the S-equol specific ELISA involves deconjugation showing high stereospecificity to S-equol without using stereospecific antibody. For the simple ELISA, we used a polyclonal antibody that targets the regions not influenced by inhibition by conjugation of glucuronate and sulfate and achieved the correlation coefficient;r = 0.975, but the value was 30 % lower than high performance liquid chromatography (HPLC). Developing upon this we invented the specific ELISA established from S-equol homogeneous combination for the standard and enzyme-labeled antigen to enhance stereospecificity. The correlation with HPLC was favorable: r = 0.986, y = 0.996x – 6. Compared to the previous method using (R,S)-equol combination, cross-reactivity with R-equol was reduced from 65 to 13 %, and that with daidzein from 0.31% to 0.08%, markedly increased in the specificity. This study is expected to be applied for both simple clinical researches, and stereospecific immunoassays in which specific antibody preparation is difficult.展开更多
文摘目的预测疥螨猪变种转录组中过敏原蛋白,对Sar s 14.3过敏原蛋白进行生物特性分析、原核表达并建立间接ELISA诊断方法。方法利用转录组测序(RNA-Seq)及BLAST、ProtParam、I-TASSER和COMPARE等软件和数据库对疥螨猪变种转录组中的过敏原组分进行预测并对疥螨Sar s 14.3过敏原蛋白序列进行理化性质分析、结构分析、同源性分析和间接ELISA诊断方法的建立。结果在疥螨猪变种的18980个转录本中共预测出390条过敏原序列,且随机匹配可能性值(E-value)为0的序列有28条。Sar s 14.3过敏原蛋白的理论相对分子质量为38 kDa,蛋白较稳定,为亲水性蛋白;二级结构以无规卷曲和β-折叠为主,其氨基酸序列与疥螨其他变种主要过敏原组分14蛋白序列相似性不低于99.76%,而与尘螨属螨虫的相似性最高为73.08%,与现行的形态学分类系统一致。所建立的间接ELISA诊断方法的最佳包被抗原浓度为2μg/mL,血清最佳稀释倍数是1∶400。结论本试验成功预测了疥螨猪变种转录组中的过敏原序列并克隆、表达了疥螨猪变种Sar s 14.3蛋白,初步预测了其分子特征,并以该过敏原蛋白为基础成功建立了一种快速的间接ELISA检测方法,可用于疥螨病的初步诊断和流行病学调查。
文摘Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary established the simple enzyme-linked immunosorbent assay (ELISA) without deconjugation, then developed the S-equol specific ELISA involves deconjugation showing high stereospecificity to S-equol without using stereospecific antibody. For the simple ELISA, we used a polyclonal antibody that targets the regions not influenced by inhibition by conjugation of glucuronate and sulfate and achieved the correlation coefficient;r = 0.975, but the value was 30 % lower than high performance liquid chromatography (HPLC). Developing upon this we invented the specific ELISA established from S-equol homogeneous combination for the standard and enzyme-labeled antigen to enhance stereospecificity. The correlation with HPLC was favorable: r = 0.986, y = 0.996x – 6. Compared to the previous method using (R,S)-equol combination, cross-reactivity with R-equol was reduced from 65 to 13 %, and that with daidzein from 0.31% to 0.08%, markedly increased in the specificity. This study is expected to be applied for both simple clinical researches, and stereospecific immunoassays in which specific antibody preparation is difficult.
