本文關(guān)鍵詞： 嘧肽霉素 煙草花葉病毒 TMV p126蛋白質(zhì) 多克隆抗體 BY-2煙草原生質(zhì)體體系 BYL體外翻譯體系　出處：《沈陽農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：嘧肽霉素(Cytosinpeptidemycin,CytPM)是新型的抗病毒藥劑,由沈陽農(nóng)業(yè)大學植物病毒研究室從鏈霉菌(Streptomyces ahygroscopicus var.liaoningensis)中提取的,該藥劑對抗病毒的防效較好,然而這種藥劑的具體分子水平上的抗病毒機制還不夠明確。本文首先用重組質(zhì)粒pET28a-HEL表達蛋白質(zhì)后并以純化的蛋白質(zhì)為抗原,制備了特異的多克隆抗體,并建立了多克隆抗體的檢測體系,最后通過Northern blot和Western blot實驗分析了嘧肽霉素對TMV的p126蛋白質(zhì)具有抑制作用。重要研究成果如下:1.成功構(gòu)建了原核表達純化載體pET28a-HEL、pET32a-HEL、pET28a-MET、pET32a-MET,將構(gòu)建好的質(zhì)粒轉(zhuǎn)化至感受態(tài)細胞BL21,經(jīng)IPTG誘導表達重組蛋白質(zhì),獲得大小分別約為25kDa、39kDa、30kDa、45kDa的重組蛋白質(zhì),與預期的蛋白質(zhì)大小一致;并發(fā)現(xiàn)重組蛋白質(zhì)pET32a-MET和pET32a-HEL經(jīng)超聲破碎離心后,沉淀處呈現(xiàn)出了大量蛋白質(zhì)條帶,上清中的蛋白質(zhì)條帶量較淺,不適于作為后續(xù)的抗原;而pET28a-HEL的蛋白質(zhì)條帶較干凈單一,故而選擇pET28a-HEL進行后續(xù)的純化實驗,該原核表達載體的成功構(gòu)建為后續(xù)多克隆抗體的制備奠定了基礎(chǔ)。2.建立了 TMV-LN抗體檢測體系,制備了 p126抗體。采用Dot-ELISA實驗檢測了抗體可用性,發(fā)現(xiàn)用CDP-Star顯色的NC膜經(jīng)過曝光可看到感染TMV的煙草葉片具有特異性斑點,而感染PMMoV的煙草葉片與健康的煙草葉片未見斑點;經(jīng)NBT、BCIP顯色反應的NC膜上接種了 TMV的煙草葉片發(fā)生變色反應,感染PMMoV的煙草葉片與健康的煙草葉片沒有發(fā)生特異性反應,說明該抗體能夠與抗原結(jié)合,表明該抗體可用。采用Western blot實驗檢測了該抗體的特異性,發(fā)現(xiàn)在接種TMV煙草葉片以及BY-2原生質(zhì)體樣品中,在130kDa附近出現(xiàn)明亮的條帶,判斷此條帶為TMV的復制蛋白質(zhì)p126,此外在180kDa處出現(xiàn)疑似p183的條帶;健康的、未感染TMV的煙草葉片樣品作為對照,沒有條帶出現(xiàn)。經(jīng)過免疫反應的PVDF膜背景干凈清晰且無雜帶,表明抗體特異性較強,用于TMV p126的檢測試驗p126多克隆抗體制備成功,為詳細研究TMV-LN的翻譯機制供給重要的研究手段。同時,也可用于詳細研究與p126互作、在病毒侵染過程中發(fā)揮重要作用的寄主蛋白質(zhì)。3.揭示了嘧肽霉素對TMV p126的表達以及核酸復制具有抑制作用。Northern blot檢測TMV RNA積累量,發(fā)現(xiàn)BY-2煙草原生質(zhì)體在用稀釋了 3200倍的嘧肽霉素處理后,原生質(zhì)體內(nèi)幾乎沒有TMV RNA正義鏈和負義鏈的積累,表明嘧肽霉素對TMV的RNA的正義鏈與負義鏈的具有很強的抑制作用;Western blot實驗檢測BY-2細胞中的TMV病毒的p126蛋白質(zhì),實驗結(jié)果表明,用稀釋3200倍嘧肽霉素處理BY-2細胞后,與TMV病毒共培養(yǎng)后沒有檢測到TMV病毒的p126復制蛋白質(zhì)及外殼蛋白質(zhì)CP,而對水處理BY-2細胞與TMV病毒共培養(yǎng)后進行Western blot檢測,發(fā)現(xiàn)在與TMV共培養(yǎng)3h后就已經(jīng)能檢測到微弱的p126復制蛋白質(zhì)條帶,隨著培養(yǎng)時間延長,檢測到的p126復制蛋白質(zhì)條帶的濃度越高,綜上說明嘧肽霉素確實能夠抑制TMV病毒的p126蛋白質(zhì)合成。此研究為深入了解嘧肽霉素對TMV病毒的分子抗病機理提供了重要參考價值。
[Abstract]:Cytosinpeptidemycin (Cytosinpeptidemycin, CytPM) is a novel antiviral agent, by the Shenyang Agricultural Uinversity chamber of plant virus (Streptomyces ahygroscopicus var.liaoningensis) from Streptomyces in the extraction of the drug against the virus prevention effect is better, but the specific antiviral mechanism of this agent on the molecular level is not clear. Firstly, the expression of pET28a-HEL protein in recombinant plasmid after taking the purified protein as antigen, polyclonal antibody was prepared, and the establishment of a detection system of polyclonal antibody, Northern blot and Western blot through the experimental analysis of the P126 protein of cytosinpeptidemycin on TMV has inhibitory effect. The important research results are as follows: 1. successfully constructed the prokaryotic expression and purification pET32a-HEL, pET28a-MET, pET28a-HEL vector, pET32a-MET, was transformed into competent cell BL21 constructed, expression induced by IPTG The recombinant protein obtained were about 25kDa, 39kDa, 30kDa, 45kDa recombinant protein, with the expected size of proteins; and found that the recombinant protein pET32a-MET and pET32a-HEL by sonication after centrifugation, precipitation showed a large number of protein bands in the supernatant of protein bands was relatively shallow, not suitable as following antigen; pET28a-HEL protein band is clean single, so pET28a-HEL was purified in subsequent experiments, the prokaryotic expression vector is successfully constructed for the subsequent preparation of polyclonal antibody of the foundation of.2. system was established to detect TMV-LN antibody, P126 antibody was prepared by Dot-ELISA. The availability of experimental antibody detection and discovery NC film by CDP-Star coloration after exposure can be seen in tobacco leaves infected by TMV with specific spots in tobacco leaves infected with PMMoV and healthy tobacco leaves no spots; The NBT NC film BCIP color reaction on tobacco leaves inoculated with the occurrence of TMV reactions, tobacco leaves infected with PMMoV and healthy tobacco leaves no specific reaction, indicating that the antibody and antigen binding, showed that the antibody can be used by Western blot. The specificity of the antibody detection, found in inoculation of TMV tobacco leaves and BY-2 protoplast samples, appeared in the vicinity of the 130kDa band bright, the bands for replication protein P126 TMV, also suspected p183 bands appeared at 180kDa; health, tobacco leaf samples were not infected with TMV as the control, no bands. After PVDF immunization the reaction is clear and clean background clutter free zone, indicates that the antibody specificity for the detection of P126 TMV test, P126 polyclonal antibody was successfully prepared, for the supply of translation mechanism study in detail the TMV-LN important research means. 鍚屾椂,涔熷彲鐢ㄤ簬璇︾粏鐮旂┒涓巔126浜掍綔,鍦ㄧ梾姣掍鏡鏌撹繃紼嬩腑鍙戞尌閲嶈浣滅敤鐨勫瘎涓昏泲鐧借川.3.鎻ず浜嗗槯鑲介湁绱犲TMV p126鐨勮〃杈句互鍙婃牳閰稿鍒跺叿鏈夋姂鍒朵綔鐢，

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