CAJ | 학술논문

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543 bp ，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20 kD。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。
목적：위료획득분진만철단백（ferritin）단백，병대해단백특정진행분석。방법근거철단백기인이지서렬，설계출상응적인물，제취분진만총RNA ，채용RT‐PCR방법확증출철단백기인，PCR산물극륭입pET32a재체，구건적중조질립pET32a‐ferritin ，경매절화측서감정후，재전화지대장간균BL21（DE3），이병기‐β‐D‐류대반유당감（IPTG）유도표체。용십이완기광산납‐취병희선알응효전영（SDS‐PAGE）감정기표체효과，용 Ni＋리자친화층석주순화기중조단백。용생물신식학방법대분진만철단백적특정진행분석。결과성공극륭분진만철단백기인병구건료 pET32a‐ferritin중조질립；분진만철단백기인개방열독광위543 bp ，편마179개안기산，PI 5．35；SDS‐PAGE결과표명철단백기인재대장간균 Bl21（DE3）중획득량호적표체，경친화층석순화후，표체산물분자질량약위20 kD。경생물신식학분석，분진만철단백함유8개사안산격매、7개소안산격매、7개락안산격매、0조안산격매린산화위점，친수구역대우소수구역，속불은정단백。결론극륭、표체료진만철단백，경순화후획득교고순도적중조단백，병대기삼급결구진행분석，위진일보연구진만과민원적결구성분급기이화성질전정이론기출。
We obtained recombinant ferritin from Dermatophagoides f arinae ,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 Bam HⅠand 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 Bl21 (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 physicochemical properties of house dust mite allergen .