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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。
Objective To clone the Der f Mal f 6 (Mal f 6) gene and construct its prokaryotic expression system. Methods The total RNA of Dust mite was extracted. According to the sequence of (AY283280.1) in GenBank, primers were designed and synthesized. The full-length Der f Mal f 6 gene was amplified by RT-PCR, cloned into pColdTF DNA vector, transformed into E. coli JM109 The positive clones were sequenced. The pCold TF-Mal f 6 plasmid was transformed into BL21 and induced by IPTG. The expressed product was verified by SDS-PAGE. The physicochemical properties, structure and function of Mal f 6 were predicted by bioinformatics software. Results The 495bp Mal f 6 gene was obtained by RT-PCR. After prokaryotic expression, SDS-PAGE electrophoresis showed that all the cells, supernatants and precipitates had protein expression, and the expression level in the supernatant was higher. Bioinformatics Analysis The protein consisted of 164 amino acids with a molecular mass of 17.7ku. The secondary structure consisted of α-helix (4.88%), extended backbone (37.8%) and random coil (57.32%), Sex cytoplasmic protein with peptidyl-prolyl trans-isomerase activity. Conclusion The prokaryotic expression of Mal f 6, an allergen of dust mite, is successful, which lays the foundation for the further research, mass production and application of this allergen.