粉尘螨变应原Der f 8全长基因克隆及表达

发布时间：2017-12-31 11:22

本文关键词：粉尘螨变应原Der f 8全长基因克隆及表达　出处：《中国寄生虫学与寄生虫病杂志》2017年04期 　论文类型：期刊论文

【摘要】：目的获得粉尘螨(Dermatophagoides farinae)变应原Der f 8全长基因并构建其原核表达质粒。方法参考Gen Bank登录号为AY283295的Der f 8部分序列设计并合成引物。以粉尘螨总RNA为模板,RT-PCR扩增获得Der f 8部分片段。采用5′cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)获得Der f 8全长序列,连接至pMD19-T载体,热转化至大肠埃希菌(Escherichia coli),挑取阳性菌落,抽提质粒后测序。根据Der f 8全长序列设计并合成引物,以粉尘螨总RNA为模板进行RT-PCR扩增,产物回收后连接pCold TF载体,热转化至E.coli,涂板过夜培养后,挑取阳性菌落,抽提质粒后测序。将pCold TF-Der f 8质粒转化至E.coli BL21,异丙基-β-D-硫代半乳糖苷诱导表达,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。采用生物信息学软件预测Der f 8的理化特性、结构和功能,并构建系统进化树。结果以Der f 8部分序列设计的引物行RT-PCR扩增获得Der f 8部分片段,长度为600 bp;5′RACE获得Der f 8剩余序列,长度为300 bp;以全长基因序列设计的引物进行RT-PCR扩增获得了Der f 8基因CDS区,长度为696 bp,测序均正确。SDSPAGE结果显示,目的蛋白为可溶性表达,相对分子质量(M_r)为81 000,与预期大小一致。序列经生物信息学分析结果显示,Der f 8全长序列与参考序列(Gen Bank登录号为AY283295)同源性为98.49%,预测其编码的疏水性蛋白具有谷胱甘肽S转移酶活性,二级结构包括α-螺旋(45.45%)、延伸主链(11.26%)和无规卷曲(43.29%)。系统进化树结果显示,粉尘螨与户尘螨(Dermatophagoides pteronyssinus)聚成一簇。结论获得了Der f 8全长基因及其原核表达质粒。
[Abstract]:Objective to obtain Dermatophagoides farinaeus. Methods the full-length gene of allergen Der f8 was constructed and its prokaryotic expression plasmid was constructed. Methods reference to Der f of Gen Bank accession number AY283295. The total RNA of Dermatophagoides farinae was used as template. The partial fragment of Der f8 was obtained by RT-PCR amplification. Rapid amplification of cDNA ends. The full-length sequence of Der f8 was obtained by race, ligated to pMD19-T vector, and heat transformed into Escherichia coli coli to pick up the positive colony. According to the full-length sequence of Der f8, primers were designed and synthesized. The total RNA of Dermatophagoides farinae was amplified by RT-PCR. The product was recovered and ligated into pCold TF vector. The heat was transformed to E. coli, and the positive colonies were picked out after overnight culture. The plasmids were extracted and sequenced. The plasmid of pCold TF-Der f8 was transformed into E. coli BL21. Isopropyl- 尾 -Dthiogalactoside induced expression. The expressed products were analyzed by SDS-PAGE. the physicochemical properties, structure and function of Der f8 were predicted by bioinformatics software. The phylogenetic tree was constructed. Results the partial Der f8 fragment was amplified by RT-PCR with the primers designed for the partial sequence of Der f8. The length of the fragment was 600 BP. The remaining sequence of Der f8 was obtained by race, the length of which was 300bp; The CDS region of the Der f8 gene was obtained by RT-PCR amplification with the primers designed for the full-length gene sequence. The length of the CDS region was 696bp, and the sequence was correct. SDS-PAGE results showed that the sequence was correct. The target protein was soluble and the relative molecular weight was 81,000, which was consistent with the expected size. The sequence was analyzed by bioinformatics. The full length sequence of Der f8 is 98.49% homology with the reference sequence, Gengen Bank login number is AY283295. It is predicted that the hydrophobic protein encodes glutathione S-transferase activity, and the secondary structure includes 伪 -helix 45.45). The results of phylogenetic tree show that the main chain is 11.26) and the random crimp is 43.29%. Dermatophagoides pteronyssinus and Dermatophagoides pteronyssinus. conclusion the full-length gene of Der f8 and its prokaryotic expression plasmid were obtained.
【作者单位】：盐城卫生职业技术学院;东南大学医学院附属盐城医院;
【基金】：国家自然科学基金(No.NSFC31272369,NSFC81001330,NSFC30060166,NSFC31572319) 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)~~
【分类号】：R384.4
【正文快照】： 尘螨与过敏性哮喘、过敏性鼻炎、特应性皮炎16、17、21、22、24类变应原的命名、理化性质,等Ⅰ~Ⅲ型变态反应性疾病密切相关。目前,临床目前尚无粉尘螨Der f 8的信息[12]。本研究采用分子使用尘螨变应原粗提浸液诊断和治疗变态反应性疾生物学技术获得了Der f 8基因全长,并用生

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