[1]马洁雪,吴乐乐,丁向真,等.在烟草叶片中瞬时表达具有生物活性的重组人纤溶酶原激活剂[J].南方医科大学学报,2019,(05):515.[doi:10.12122/j.issn.1673-4254.2019.05.03]
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在烟草叶片中瞬时表达具有生物活性的重组人纤溶酶原激活剂()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2019年05期
页码:
515
栏目:
出版日期:
2019-05-15

文章信息/Info

Title:
Transient expression of bioactive recombinanthumanplasminogen activator in tobacco leaf
作者:
马洁雪吴乐乐丁向真李志英王盛
关键词:
烟草花叶病毒Zera标签重组人纤溶酶原激活剂表达烟草
Keywords:
tobacco mosaic virus Zera Tags recombinant human plasminogen activator expression tobacco
DOI:
10.12122/j.issn.1673-4254.2019.05.03
摘要:
目的探索在植物中瞬时高效表达重组人纤溶酶原激活剂(rhPA)的可行性,探寻低成本rhPA表达新途径。方法构建表 达rhPA的烟草花叶病毒(TMV)表达载体pTMV rhPA-NSK和Zera融合标签植物表达载体pJ Zera-rhPA;农杆菌渗滤接种受体 植物Nicotiana benthamiana和N. excelsiana叶片;观察叶片接种部位表型变化;Western blot和ELISA测定和分析rhPA在受体 烟草中的表达情况及表达产物特点;纤维蛋白琼脂糖平板法评价rhPA的体外生物活性。结果病毒载体实验结果显示,pTMV rhPA-NSK病毒载体在N. benthamiana和N. excelsiana叶片接种部位产生不同程度的坏死症状;在坏死组织中依然存在完整的 rhPA蛋白;N. benthamiana中rhPA的表达量略高于N. excelsiana(P<0.05),约占接种叶片可溶性总蛋白的0.6%;表达的rhPA具 有体外溶栓活性。Zera融合标签实验结果显示,pJ Zera-rhPA表达载体在烟草叶片接种部位未造成坏死;Zera标签与其融合蛋 白rhPA之间可能存在着易断裂位点;Zera标签蛋白微粒的形成是一种逐步积累聚集的过程;在微粒形成过程中,部分可溶性多 肽可能被包裹进了微粒。结论在烟草中可以快速地表达具有生物学活性的rhPA,植物病毒表达系统为低成本rhPA的生产提 供一种富有潜力的新平台。
Abstract:
Objective To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. Methods Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). Results Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P<0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. Conclusion Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.
更新日期/Last Update: 1900-01-01