CAJ | 학술논문

[目的] 研究了植物病毒表达载体pClYVV/CP/W的构建及用pClYVV/CP/W表达绿色荧光蛋白(GFP),为植物反应器生产有用蛋白、开发有效的植物病毒载体提供参考.[方法] 用三叶草黄脉病毒侵染性全长cDNA 克隆pClYVV基因组的Nib/CP基因之间的一段多克隆位点和两侧含有病毒蛋白酶Nia切割识别序列的多聚脱氧核糖核苷酸接头,构建pClYVV/CP/W载体,并将绿色荧光蛋白基因插入pClYVV/CP/W中构建pClYVV/CP/W/GFP载体,用反转录PCR对重组病毒克隆转录情况进行检测,并用Western杂交对重组病毒克隆表达的目的基因产物进行测定.[结果] pClYVV/CP/W/GFP接种的蚕豆幼苗表现出与野生型ClYVV相同的症状,发病率达100%,表明重组病毒克隆pClYVV/CP/W/GFP有侵染性,外源基因的插入没有破坏载体pClYVV/CP/W的开放阅读框；以感染叶总RNA为模板,对pClYVV/CP/W/GFP表达GFP外源基因的稳定性进行了检测,从F0 (重组病毒质粒最初转录出的病毒)一直检测到F4子代病毒.检测结果表明,外源基因在F4子代病毒基因组中稳定存在；以从感染叶中提取的总蛋白为抗原,以GFP抗体为第1抗体,以碱性磷酸酶标记抗体为第2抗体,用Western杂交对重组病毒克隆pCVYVV/CP/W/GFP表达外源蛋白进行了定性检测.从F0一直检测到F4子代病毒.检测结果表明,重组病毒克隆pClYVV/CP/W/GFP至少到F4子代病毒能稳定表达GFP.[结论] 该研究结果为用植物表达有用蛋白提供了有用的植物病毒载体.
[목적] 연구료식물병독표체재체pClYVV/CP/W적구건급용pClYVV/CP/W표체록색형광단백(GFP),위식물반응기생산유용단백、개발유효적식물병독재체제공삼고.[방법] 용삼협초황맥병독침염성전장cDNA 극륭pClYVV기인조적Nib/CP기인지간적일단다극륭위점화량측함유병독단백매Nia절할식별서렬적다취탈양핵당핵감산접두,구건pClYVV/CP/W재체,병장록색형광단백기인삽입pClYVV/CP/W중구건pClYVV/CP/W/GFP재체,용반전록PCR대중조병독극륭전록정황진행검측,병용Western잡교대중조병독극륭표체적목적기인산물진행측정.[결과] pClYVV/CP/W/GFP접충적잠두유묘표현출여야생형ClYVV상동적증상,발병솔체100%,표명중조병독극륭pClYVV/CP/W/GFP유침염성,외원기인적삽입몰유파배재체pClYVV/CP/W적개방열독광；이감염협총RNA위모판,대pClYVV/CP/W/GFP표체GFP외원기인적은정성진행료검측,종F0 (중조병독질립최초전록출적병독)일직검측도F4자대병독.검측결과표명,외원기인재F4자대병독기인조중은정존재；이종감염협중제취적총단백위항원,이GFP항체위제1항체,이감성린산매표기항체위제2항체,용Western잡교대중조병독극륭pCVYVV/CP/W/GFP표체외원단백진행료정성검측.종F0일직검측도F4자대병독.검측결과표명,중조병독극륭pClYVV/CP/W/GFP지소도F4자대병독능은정표체GFP.[결론] 해연구결과위용식물표체유용단백제공료유용적식물병독재체.
[Objective] The study was to report the construction of plant virus expression vector pClYVV/CP/W and the expression of green fluorescent protein(GFP) with pClYVV/CP/W, and to develop effective plat virus vector for plant bioreactor to produce useful protein.[Method] A section of multiple cloning sites among NIb/CP genes in pClYVV genome and deoxyribonucleotide polylinker of cleavage recognition sequence containing viral protease NIa were cloned with infectivity full-length cDNA of clover yellow vein virus (ClYVV), and pClYVV/CP/W vector was constructed, GFP gene was inserted into pClYVV/CP/W to construct the pClYVV/CP/W/GFP vector. The transcription situation of recombinant virus clone was detected by RT-PCR, and targeted gene products expressed by recombinant virus clone were detected with western blot (WB). [Result] The broad bean seedling inoculated with pClYVV/CP/W/GFP expressed the same symptom as wild type ClYVV, morbidity was of 100%, the result showed that recombinant virus clone pClYVV/CP/W/GFP didn't suppress, insertion of foreign gene didn't destroy the open reading frame of pClYVV/CP/W. Foreign gene can keep living in F4 progeny virus genome steadily, recombinant virus clone pClYVV/CP/W/GFP could steadily express GFP in progeny virus at least. [Conclusion] The useful plant virus vector was provided for useful protein expressing.