發(fā)布時(shí)間：2017-12-31 11:22

  本文關(guān)鍵詞：粉塵螨變應(yīng)原Der f 8全長(zhǎng)基因克隆及表達(dá)　出處：《中國(guó)寄生蟲(chóng)學(xué)與寄生蟲(chóng)病雜志》2017年04期 　論文類(lèi)型：期刊論文

  更多相關(guān)文章： 粉塵螨 變應(yīng)原 基因克隆 基因表達(dá) 生物信息學(xué)

【摘要】：目的獲得粉塵螨(Dermatophagoides farinae)變應(yīng)原Der f 8全長(zhǎng)基因并構(gòu)建其原核表達(dá)質(zhì)粒。方法參考Gen Bank登錄號(hào)為AY283295的Der f 8部分序列設(shè)計(jì)并合成引物。以粉塵螨總RNA為模板,RT-PCR擴(kuò)增獲得Der f 8部分片段。采用5′cDNA末端快速擴(kuò)增技術(shù)(rapid amplification of cDNA ends,RACE)獲得Der f 8全長(zhǎng)序列,連接至pMD19-T載體,熱轉(zhuǎn)化至大腸埃希菌(Escherichia coli),挑取陽(yáng)性菌落,抽提質(zhì)粒后測(cè)序。根據(jù)Der f 8全長(zhǎng)序列設(shè)計(jì)并合成引物,以粉塵螨總RNA為模板進(jìn)行RT-PCR擴(kuò)增,產(chǎn)物回收后連接pCold TF載體,熱轉(zhuǎn)化至E.coli,涂板過(guò)夜培養(yǎng)后,挑取陽(yáng)性菌落,抽提質(zhì)粒后測(cè)序。將pCold TF-Der f 8質(zhì)粒轉(zhuǎn)化至E.coli BL21,異丙基-β-D-硫代半乳糖苷誘導(dǎo)表達(dá),采用十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析表達(dá)產(chǎn)物。采用生物信息學(xué)軟件預(yù)測(cè)Der f 8的理化特性、結(jié)構(gòu)和功能,并構(gòu)建系統(tǒng)進(jìn)化樹(shù)。結(jié)果以Der f 8部分序列設(shè)計(jì)的引物行RT-PCR擴(kuò)增獲得Der f 8部分片段,長(zhǎng)度為600 bp;5′RACE獲得Der f 8剩余序列,長(zhǎng)度為300 bp;以全長(zhǎng)基因序列設(shè)計(jì)的引物進(jìn)行RT-PCR擴(kuò)增獲得了Der f 8基因CDS區(qū),長(zhǎng)度為696 bp,測(cè)序均正確。SDSPAGE結(jié)果顯示,目的蛋白為可溶性表達(dá),相對(duì)分子質(zhì)量(M_r)為81 000,與預(yù)期大小一致。序列經(jīng)生物信息學(xué)分析結(jié)果顯示,Der f 8全長(zhǎng)序列與參考序列(Gen Bank登錄號(hào)為AY283295)同源性為98.49%,預(yù)測(cè)其編碼的疏水性蛋白具有谷胱甘肽S轉(zhuǎn)移酶活性,二級(jí)結(jié)構(gòu)包括α-螺旋(45.45%)、延伸主鏈(11.26%)和無(wú)規(guī)卷曲(43.29%)。系統(tǒng)進(jìn)化樹(shù)結(jié)果顯示,粉塵螨與戶(hù)塵螨(Dermatophagoides pteronyssinus)聚成一簇。結(jié)論獲得了Der f 8全長(zhǎng)基因及其原核表達(dá)質(zhì)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 偽 -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.
【作者單位】： 鹽城衛(wèi)生職業(yè)技術(shù)學(xué)院;東南大學(xué)醫(yī)學(xué)院附屬鹽城醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金(No.NSFC31272369,NSFC81001330,NSFC30060166,NSFC31572319) 江蘇省衛(wèi)生廳招標(biāo)項(xiàng)目(No.Z200914) 江蘇省衛(wèi)生職業(yè)技術(shù)教育研究立項(xiàng)課題(No.J200907)~~
【分類(lèi)號(hào)】：R384.4
【正文快照】： 塵螨與過(guò)敏性哮喘、過(guò)敏性鼻炎、特應(yīng)性皮炎16、17、21、22、24類(lèi)變應(yīng)原的命名、理化性質(zhì),等Ⅰ~Ⅲ型變態(tài)反應(yīng)性疾病密切相關(guān)。目前,臨床目前尚無(wú)粉塵螨Der f 8的信息[12]。本研究采用分子使用塵螨變應(yīng)原粗提浸液診斷和治療變態(tài)反應(yīng)性疾生物學(xué)技術(shù)獲得了Der f 8基因全長(zhǎng),并用生

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