粉尘螨变应原Der f Mal f 6的克

发布时间：2018-06-20 09:39

本文选题：粉尘螨 + 变应原　；参考：《中国病原生物学杂志》2013年06期

【摘要】：目的克隆粉尘螨变应原Der f Mal f 6(简称Mal f 6)编码基因并构建其原核表达体系。方法提取粉尘螨总RNA,根据GenBank中的(AY283280.1)序列设计并合成引物,RT-PCR扩增Der f Mal f 6全长基因,克隆至pColdTF DNA载体,转化至E.coli JM109,取阳性克隆测序;将pCold TF-Mal f 6质粒转化至BL21,IPTG诱导表达,采用SDS-PAGE验证表达产物,采用生物信息学软件预测Mal f 6的理化特性、结构和功能。结果 RT-PCR获得全长为495bp的Mal f 6基因。原核表达后经SDS-PAGE电泳显示其全细胞、上清及沉淀物均有蛋白表达,以上清表达量较高。生物信息学分析该蛋白由164个氨基酸组成,分子质量单位为17.7ku,二级结构由α螺旋(4.88%)、延伸主链(37.8%)和无规则卷曲(57.32%)组成,是亲水性细胞质蛋白,具有肽酰-脯氨酰反式异构酶活性。结论粉尘螨变应原Mal f 6原核表达获得成功,为该变应原的进一步研究及规模生产和应用奠定了基础。
[Abstract]:Objective to clone the gene encoding the allergen Der f Mal f 6 of Dermatophagoides farinae and construct its prokaryotic expression system. Methods the total RNAs of Dermatophagoides farinae were extracted. The full-length gene of Der f Mal f 6 was amplified by RT-PCR according to the sequence of AY283280.1 in GenBank, cloned into pColdTF DNA vector, transformed into E. coli JM109, and sequenced, and the plasmid pCold TF-Mal f 6 was transformed into BL21 IPTG. SDS-PAGE was used to verify the expressed product and bioinformatics software was used to predict the physical and chemical properties, structure and function of Mal f 6. Results the full length of Mal f 6 gene of 495bp was obtained by reverse transcription-polymerase chain reaction (RT-PCR). After prokaryotic expression, SDS-PAGE electrophoresis showed that protein was expressed in the whole cell, supernatant and sediment. Bioinformatics analysis showed that the protein was composed of 164 amino acids with a molecular weight unit of 17.7 ku.The secondary structure consisted of a helix 4.88a, an extension of the main chain 37.8% and an irregular crimp of 57.32). It was a hydrophilic cytoplasmic protein with peptidyl-proline reverse isomerase activity. Conclusion the prokaryotic expression of the allergen Mal f 6 of Dermatophagoides farinae was successful, which laid a foundation for the further study, scale production and application of the allergen.
【作者单位】：盐城卫生职业技术学院;
【基金】：国家自然科学基金项目(No.NS-FC31272369,NS-FC81001330) 江苏省卫生厅招标项目(No.Z200914) 江苏省卫生职业技术教育研究立项课题(No.J200907) 江苏省“六大人才高峰”第六批资助项目
【分类号】：R392

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