粉尘螨铁蛋白的克隆、表达及蛋白特征分析

Cloning,expression and purification of dust mite ferritin and its molecular characteristics

目的为了获得粉尘螨铁蛋白(ferritin)蛋白,并对该蛋白特征进行分析。方法根据铁蛋白基因已知序列,设计出相应的引物,提取粉尘螨总RNA,采用RT-PCR方法扩增出铁蛋白基因,PCR产物克隆入pET32a载体,构建的重组质粒pET32a-ferritin,经酶切和测序鉴定后,再转化至大肠杆菌BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定其表达效果,用Ni+离子亲和层析柱纯化其重组蛋白。用生物信息学方法对粉尘螨铁蛋白的特征进行分析。结果成功克隆粉尘螨铁蛋白基因并构建了pET32a-ferritin重组质粒;粉尘螨铁蛋白基因开放阅读框为543bp,编码179个氨基酸,PI 5.35;SDS-PAGE结果表明铁蛋白基因在大肠杆菌Bl21(DE3)中获得良好的表达,经亲和层析纯化后,表达产物分子质量约为20kD。经生物信息学分析,粉尘螨铁蛋白含有8个丝氨酸激酶、7个苏氨酸激酶、7个酪氨酸激酶、0组氨酸激酶磷酸化位点,亲水区域大于疏水区域,属不稳定蛋白。结论克隆、表达了尘螨铁蛋白,经纯化后获得较高纯度的重组蛋白,并对其三级结构进行分析,为进一步研究尘螨过敏原的结构成分及其理化性质奠定理论基础。

We obtained recombinant ferritin from Dermatophagoides farinae, and analyzed the characterization of the pro- tein. A pair of primers was designed according to the known sequence of ferritin gene. The live mites identified and cultured lo- cally were picked and the total RNA was extracted. The ferritin gene fragment was amplified by RT-PCR, and cloned into pET32a vector, and then transferred into E. coli Top10. The target gene obtained from the recombinant plasmid by digestion with Barn H I and Hind Ⅲ was connected to the prokaryotic expression vector pET-32a. The expressed recombinant plasmid containing ferritin gene was constructed by cloning target gene into pET-32a and transferred into E. coli B121 （DE3）. The ex- pressed recombinant protein was analyzed by SDS-PAGE, and was purified by immobilized metal ion affinity chromatography （IMAC）. The ferritin expressed by dust mite was analyzed by the method of bioinformatics. The recombinant plasmid pET32a- ferritin was constructed. SDS-PAGE showed a correct molecular weight of the recombinant ferritin protein. After purification by affinity chromatography, the protein showed only one strip on SDS-PAGE gel. SDS-PAGE showed a band at 20 kD. Dust mite ferritin contains 8 serine kinase, 7 threonine kinase, 7 tyrosine kinase, and 0 histidine kinase phosphorylation sites. Hy- drophilic region is larger than the hydrophobic region and it is an unstable protein. In conclusion, the ferritin gene has been cloned and expressed. The purified ferritin has high purity. The study provides a basis for further study of composition and physieoehemical properties of house dust mite allergen.