嘧肽霉素对烟草花叶病毒蛋白质表达作用机制研究

发布时间：2018-02-04 19:43

  本文关键词： 嘧肽霉素 烟草花叶病毒 TMV p126蛋白质 多克隆抗体 BY-2烟草原生质体体系 BYL体外翻译体系　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：嘧肽霉素(Cytosinpeptidemycin,CytPM)是新型的抗病毒药剂,由沈阳农业大学植物病毒研究室从链霉菌(Streptomyces ahygroscopicus var.liaoningensis)中提取的,该药剂对抗病毒的防效较好,然而这种药剂的具体分子水平上的抗病毒机制还不够明确。本文首先用重组质粒pET28a-HEL表达蛋白质后并以纯化的蛋白质为抗原,制备了特异的多克隆抗体,并建立了多克隆抗体的检测体系,最后通过Northern blot和Western blot实验分析了嘧肽霉素对TMV的p126蛋白质具有抑制作用。重要研究成果如下:1.成功构建了原核表达纯化载体pET28a-HEL、pET32a-HEL、pET28a-MET、pET32a-MET,将构建好的质粒转化至感受态细胞BL21,经IPTG诱导表达重组蛋白质,获得大小分别约为25kDa、39kDa、30kDa、45kDa的重组蛋白质,与预期的蛋白质大小一致;并发现重组蛋白质pET32a-MET和pET32a-HEL经超声破碎离心后,沉淀处呈现出了大量蛋白质条带,上清中的蛋白质条带量较浅,不适于作为后续的抗原;而pET28a-HEL的蛋白质条带较干净单一,故而选择pET28a-HEL进行后续的纯化实验,该原核表达载体的成功构建为后续多克隆抗体的制备奠定了基础。2.建立了 TMV-LN抗体检测体系,制备了 p126抗体。采用Dot-ELISA实验检测了抗体可用性,发现用CDP-Star显色的NC膜经过曝光可看到感染TMV的烟草叶片具有特异性斑点,而感染PMMoV的烟草叶片与健康的烟草叶片未见斑点;经NBT、BCIP显色反应的NC膜上接种了 TMV的烟草叶片发生变色反应,感染PMMoV的烟草叶片与健康的烟草叶片没有发生特异性反应,说明该抗体能够与抗原结合,表明该抗体可用。采用Western blot实验检测了该抗体的特异性,发现在接种TMV烟草叶片以及BY-2原生质体样品中,在130kDa附近出现明亮的条带,判断此条带为TMV的复制蛋白质p126,此外在180kDa处出现疑似p183的条带;健康的、未感染TMV的烟草叶片样品作为对照,没有条带出现。经过免疫反应的PVDF膜背景干净清晰且无杂带,表明抗体特异性较强,用于TMV p126的检测试验p126多克隆抗体制备成功,为详细研究TMV-LN的翻译机制供给重要的研究手段。同时,也可用于详细研究与p126互作、在病毒侵染过程中发挥重要作用的寄主蛋白质。3.揭示了嘧肽霉素对TMV p126的表达以及核酸复制具有抑制作用。Northern blot检测TMV RNA积累量,发现BY-2烟草原生质体在用稀释了 3200倍的嘧肽霉素处理后,原生质体内几乎没有TMV RNA正义链和负义链的积累,表明嘧肽霉素对TMV的RNA的正义链与负义链的具有很强的抑制作用;Western blot实验检测BY-2细胞中的TMV病毒的p126蛋白质,实验结果表明,用稀释3200倍嘧肽霉素处理BY-2细胞后,与TMV病毒共培养后没有检测到TMV病毒的p126复制蛋白质及外壳蛋白质CP,而对水处理BY-2细胞与TMV病毒共培养后进行Western blot检测,发现在与TMV共培养3h后就已经能检测到微弱的p126复制蛋白质条带,随着培养时间延长,检测到的p126复制蛋白质条带的浓度越高,综上说明嘧肽霉素确实能够抑制TMV病毒的p126蛋白质合成。此研究为深入了解嘧肽霉素对TMV病毒的分子抗病机理提供了重要参考价值。
[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梾姣掍镜鏌撹繃绋嬩腑鍙戞尌閲嶈�佷綔鐢ㄧ殑瀵勪富铔嬬櫧璐�.3.鎻�绀轰簡鍢ц偨闇夌礌瀵筎MV p126鐨勮〃杈句互鍙婃牳閰稿�嶅埗鍏锋湁鎶戝埗浣滅敚�

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