疥螨Sar s 14.3过敏原蛋白原核表达及间接ELISA方法的建立

Prokaryotic expression of Sar s 14.3 allergen from Sarcoptes scabiei and establishment of an indirect ELISA method

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。

This study was aimed at predicting the allergens of the Sarcoptes scabiei var.suis transcriptome and the molecular characteristics of the Sar s 14.3 allergen,and to assess whether the protein might serve as a target diagnostic marker for Sarcoptes scabiei var.suis.RNA-Seq technology,bioinformatics software and databases including BLAST,ProtParam,I-TASSER and COMPARE were used to predict the allergen components in the transcriptome,the physicochemical properties,the molecular structure characteristics and homologous proteins of the Sar s 14.3 allergen.A total of 18980 transcripts were identified,390 of which were predicted to encode allergens,including the Sar s 14.3 protein coding gene.The Sar s 14.3 allergen was predicted to be a hydrophilic protein of 38 kDa,and secondary structure analysis revealed that it contained mainlyα-helices and coils.According to BLAST results,the similarity of the amino acid sequences of group 14 allergens within the genus Sarcoptes exceeded 99.7%but was lower with respect to the genus Dermatophagoides,in agreement with with morphologic taxonomy.Moreover,the established indirect ELISA diagnostic method indicated that the optimal concentration of coating antigen was 2μg/mL,and the optimal dilution ratio of serum was 1∶400.In conclusion,allergen proteins in the transcriptome of Sarcoptes scabiei var.suis were successfully predicted;the Sar s 14.3 protein was cloned and expressed;and a rapid,efficient and accurate indirect ELISA diagnostic method was successfully established.