利用烟草表达重组牛凝乳酶及人表皮生长因子的研究

发布时间：2018-11-26 06:42

【摘要】：植物生物反应器以植物细胞为载体,利用植物细胞的各种组分来合成人类需要的产品。相较于其他生物反应器,植物生物反应器具有综合成本低、产品安全性高、生产易于规模化等优点,现已广泛应用于工业用蛋白(酶)和医药蛋白的生产。本研究以转基因烟草植株作为植物生物反应器,对在烟草叶片中表达的重组牛凝乳酶和重组人表皮细胞生长因子进行了探索和尝试,获得以下研究成果：构建了牛凝乳酶原和前牛凝乳酶原基因的植物细胞核转化载体四个,分别为p33cym11、p33cyml2、p33cym21和p33cym22。这四个载体中,(前)牛凝乳原基因由于不同的信号肽而有所区别,重组凝乳酶的羧基端(C端)融合有6×组氨酸标签,目的是为了表达后通过亲和层析分离重组凝乳酶提供方便。通过农杆菌介导法,把上述基因导入烟草,经过草甘膦筛选,获得了抗性再生植株；经过PCR和Southern杂交检测,证实抗性再生植株中(前)牛凝乳酶原基因已经整合到基因组中,经RT-PCR检测,证明了外源基因在烟草细胞中得到转录。利用金标免疫试纸条检测,证明了转基因烟草中标记基因得到了表达；Western检测表明,重组牛凝乳酶已经得到表达；经ELISA实验表明其表达量占总可溶性蛋白的比例在0.12%～0.52%之间,也就是35.2～83.2 ng/g鲜重；凝乳实验表明,重组凝乳酶具有良好的凝乳活性,与市场上的产品活性相当。构建了人表皮生长因子基因的烟草叶绿体转化表达载体pWX-Nt02。该载体中含有报告基因smGFP、选择标记基因aadA以及目的基因hEGF;载体中目的基因下游添加了6×组氨酸标签序列,便于目的蛋白的分离纯化。通过基因枪法将载体pWX-Nt02导入烟草叶绿体基因组中,经过筛选,获得了转基因植株。结合分子检测和报告基因表达分析,经过4轮筛选,获得了同质化的叶绿体转基因烟草植株。经过PCR和Southern杂交分析,证实外源基因已插入烟草叶绿体基因组中,并且达到同质化。同质化的转基因烟草有很好的遗传稳定性,严格地遵循母性遗传特性。荧光分析和SDS-PAGE分析证明,报告基因绿色荧光蛋白(GFP)成功表达；ELISA检测表明,成熟叶片中GFP的表达量占总可溶性蛋白的22.38%,即GFP的积累量达到4.22 mg/g鲜重；通过硫酸铵分级沉淀和凝胶过滤层析的方法,分离纯化了绿色荧光蛋白。Western检测证实目的蛋白重组人表皮生长因子已经得到表达。ELISA实验表明,其表达量为可溶性蛋白的0.124%～0.165%,即23.16～25.77 ng/g鲜重。细胞增值实验和延伸实验表明,烟草表达的重组人表皮生长因子具有很好的生物活性。
[Abstract]:Plant bioreactor uses plant cells as carrier and uses various components of plant cells to synthesize products needed by human beings. Compared with other bioreactors, plant bioreactor has many advantages, such as low cost, high product safety, easy to scale production, and has been widely used in the production of industrial protein (enzyme) and pharmaceutical protein. In this study, transgenic tobacco plants were used as plant bioreactor to explore the expression of recombinant bovine condensate enzyme and recombinant human epidermal growth factor in tobacco leaves. The following results were obtained: four plant nuclear transformation vectors of bovine prothrombin gene and procoagulinogen gene were constructed, which were p33cym11p33cyml2p33cym21 and p33cym22, respectively. Among the four vectors, the bovine procoagulant gene is different because of different signal peptides, and the carboxyl terminal (C-terminal) of the recombinant coagulase has a 6 脳 histidine label. The aim of this study was to provide convenience for the separation of recombinant curd by affinity chromatography. The above genes were introduced into tobacco by Agrobacterium tumefaciens, and the resistant regenerated plants were obtained by glyphosate screening. The results of PCR and Southern hybridization showed that bovine procoagulase genes in resistant regenerated plants had been integrated into the genome, and that exogenous genes were transcribed in tobacco cells by RT-PCR detection. The expression of marker gene in transgenic tobacco was proved by using gold labeled immunosorbent strip test, and the expression of recombinant bovine curd enzyme was confirmed by Western assay. ELISA test showed that the proportion of the total soluble protein was 0.12%, that is, the fresh weight of 35.2% (83.2 ng/g). The result of curd experiment showed that the recombinant coagulase had good curd activity, which was equivalent to the product activity in the market. The expression vector pWX-Nt02. of tobacco chloroplast transformation of human epidermal growth factor gene was constructed. The reporter gene smGFP, selective marker gene aadA and the downstream target gene 6 脳 histidine label sequence were added to the vector of the target gene hEGF;, which was convenient for the separation and purification of the target protein. The vector pWX-Nt02 was introduced into the chloroplast genome of tobacco by gene gun method, and the transgenic plants were obtained by screening. After four rounds of screening, homogenized chloroplast transgenic tobacco plants were obtained by molecular detection and reporter gene expression analysis. By PCR and Southern hybridization analysis, it was confirmed that the exogenous gene had been inserted into the chloroplast genome of tobacco and reached homogenization. Homogenized transgenic tobacco has good genetic stability and strictly follows maternal genetic characteristics. Fluorescence analysis and SDS-PAGE analysis showed that the reporter gene green fluorescent protein (GFP) was successfully expressed, and ELISA analysis showed that the expression of GFP in mature leaves accounted for 22.38% of the total soluble protein, that is, the accumulation of GFP reached 4.22 mg/g fresh weight. Green fluorescent protein was isolated and purified by ammonium sulfate fractionation and gel filtration chromatography. Western assay confirmed that the recombinant human epidermal growth factor (EGF) of the target protein had been expressed. ELISA assay showed that the recombinant human epidermal growth factor (EGF) was expressed. Its expression was 0.124% of the soluble protein 0.165, that is 23.16525.77 ng/g fresh weight. Cell proliferation and extension experiments showed that recombinant human epidermal growth factor (HEGF) expressed by tobacco had good biological activity.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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