| 巫娟 张俊艳 何颖 陈惠芳 李碧舟 关志澳 陶爱林.粉尘螨过敏原蛋白Der f 3 的表达纯化及活性鉴定[J].,2016,16(1):54-57 |
| 粉尘螨过敏原蛋白Der f 3 的表达纯化及活性鉴定 |
| Expression, Purification and Biological Activity Identification of House DustMite Allergen Der f 3 |
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| DOI： |
| 中文关键词: Der f 3 复性 酶学活性 免疫学活性 |
| 英文关键词: Der f 3 Renaturation Enzymatic activity Immunological activity |
| 基金项目:国家重大科技专项重大课题（2014ZX08011-005）；国家重大科技专项重点课题（2014ZX08011-005B）；
国家自然科学基金项目（81373128） |
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| 中文摘要: |
| 目的：制备有生物学活性的粉尘螨过敏原蛋白Der f 3。方法：构建带有Strep Ⅱ标签的原核表达体系Rosetta-pET44a-Der f 3
高效表达目的蛋白，分别用梯度透析法和亲和层析法对其进行复性纯化，通过Western Blot 试验验证目的蛋白的有效表达，最后
用丝氨酸蛋白酶特异荧光底物及粉尘螨过敏病人血清分别鉴定目的蛋白的酶学活性及免疫学活性。结果：利用原核表达体系成
功表达了目的蛋白Der f 3，Western Blot 结果显示有目的条带出现。复性纯化后的过敏原蛋白Der f 3能够成功降解丝氨酸蛋白酶
特异性荧光底物，ELISA 试验结果显示该蛋白血清特异性IgE 在4 级以上粉尘螨过敏病人中阳性率为4 %。结论：成功获得有酶
学活性及免疫学活性的粉尘螨过敏原蛋白Der f 3，为生产高质量的诊断试剂和疫苗奠定了基础。 |
| 英文摘要: |
| Objective:To obtain the biologically active house dust mite allergen Der f 3.Methods:The expression vector
pET44a-Der f 3 contained Strep tag Ⅱ for producing target protein was constructed efficiently. Recombinant protein was renatured by
gradient dialysis, analysed by Western Blot and purified by affinity chromatography. The enzymatic activity of Der f 3 was identified by
using specific fluorogenic substrates. Plasma from house dust mite allergic patients was used to evaluate immunological activity of Der f
3.Results:The target protein was successfully expressed in prokaryotic expression system. The target band appeared as expected on
Western Blot profile. Specific substrates broke down successfully by the action of the actived and purified house dust mite allergen Der f
3. The results of the enzyme-linked immunosorbent assay showed 4 %positive rate of the specific antibody IgE among the plasma of patients
suffering from allergy against house dust mite.Conclusion:The house dust mite allergen Der f 3 was expressed successfully with
enzymatic activity and immunological activity. So it could be used as diagnostic reagent and vaccine of high quality. |
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