【作者】 李静；

【导师】 沈法富；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 随着分子生物学和遗传转化技术的发展,利用转基因植物生产人或动物基因工程药物蛋白已成为植物基因工程的一个新兴研究领域。与目前的细菌、酵母及哺乳动物细胞等传统药物蛋白生产系统相比,用转基因植物生产基因工程药物蛋白具有其独特的优势:成本低,可以以农业化的规模生产昂贵的生化药物;完整的真核细胞表达系统,使表达产物具有较好的生物活性;安全性好,无外源性病原污染;口服植物疫苗能诱导粘膜免疫反应,生产简便、成本低廉,不需要冷藏和低温运输。但植物表达存在表达量低、下游分离纯化困难等严重问题,因此寻找新的转基因植物受体,开发新的植物表达系统,成为植物生物反应器研究的重点。本研究以叶用生菜(Lactuca sativa L.sp)为试验材料,探讨优化生菜农杆菌真空渗透瞬时转化方法和稳定转化组织培养体系,采用瞬时转化方法将四种不同的干扰素-β基因转入生菜中,研究生菜瞬时表达干扰素-β的水平和生物活性,获得结果如下:1.对生菜的瞬时表达方法进行了系统优化,建立了适宜生菜高效瞬时表达的转化体系(200μmol/L乙酰丁香酮、0.8OD600菌液密度、真空处理30min、共培养6d)。以条件A1B1C1D1(0μmol/L乙酰丁香酮、0.4OD600菌液密度、真空处理10min、共培养2d)为对照,应用结果表明:优势组合的GUS表达水平为21.39nmol? mg-1? min-1 MU,比对照(1.31 nmol? mg-1? min-1 MU)提高16.3倍。结果表明该生菜瞬时表达体系可有效的提高外源基因的表达。2.糖蛋白药物干扰素-β在生菜叶片中成功表达为27kDa的蛋白,推测为带有植物特有糖基化形式的糖蛋白;抗病毒活性表明,该干扰素-β能抑制水泡性口膜炎病毒对人羊膜Wish细胞的攻击,这也是首次对农杆菌真空渗透瞬时表达来源的生化药物进行生物活性检测的报道。由于基因定点突变使第17位半胱氨酸改为丝氨酸(Cys-Ser),去除了Cys17对正常二硫键(Cys31和Cys141)的影响,使干扰素β的空间结构更稳定,因此,突变型的干扰素表达量和活性高于原始型干扰素。四种干扰素-β(IFN,无信号肽的干扰素-β;sIFN,有信号肽的干扰素-β;mIFN,无信号肽突变型的干扰素-β;smIFN,有信号肽突变型的干扰素-β)表达产物的活性分别为:3.1×104IU/mL,5.8×104IU/mL,6.3×104IU/mL,9.8×104IU/mL;表达量也依次升高。表明:Cys17向Ser17的改变及信号肽的添加有利于干扰素-β表达量和活性的提高,可以利用农杆菌介导的瞬时表达方法快速、大量、廉价的生产人源细胞因子、疫苗等生化药物。3.为探讨干扰素-β在植物中的糖基化形式,构建含有糖基酶PNGase F的植物双元表达载体pBI121-F,将含有pBI121-F的农杆菌GV3101与含有pBI121-smIFN的农杆菌GV3101混合后,真空渗透转化生菜叶片,以期利用PNGase F的酰胺内切酶作用将干扰素-β的糖链切除,但通过Western检测,没有发现干扰素-β分子量的变化,可能是由于干扰素-β的表达量较低,不能检测到PNGase F的作用,也可能是由于植物糖基化结构中含有较多的α-1,3-岩藻糖,α-1,3-岩藻糖的位阻影响了PNGase F的酶切作用。4.以三种不同基因型生菜(日本生菜,美国大叶速生,泰国生菜)的两种外植体(10d龄的子叶和真叶)为材料,建立了适合多种基因型生菜组织培养和植株再生体系:MS培养基添加0.1 mg/L NAA和0.1-0.5mg/L 6-BA,为不同基因型生菜的最适不定芽诱导培养基;基因型不同,所要求的激素浓度配比也不同:日本生菜的高效不定芽诱导激素配比为0.1mg/L 6-BA和0.1mg/L NAA,美国大叶速生为0.3mg/L 6-BA and 0.1mg/L NAA,泰国生菜为0.5mg/L 6-BA和0.1mg/L NAA;真叶的不定芽分化率显著高于子叶的分化率。最适的生根培养基为添加0.1mg/L NAA的MS;生菜不定芽诱导生根较易,基因型和外植体类型对生根的影响较小;基因转化中卡那霉素的适宜筛选浓度为50mg/L。生菜为强自花授粉作物,转入基因不容易丢失;生菜叶片可直接生食。该体系的建立有利于生菜的基因转化,对利用生菜稳定表达基因工程类药物奠定试验基础。5.采用重叠延伸PCR的方法构建了与GUS基因融合的IFN基因片段,以期利用GUS的高表达带动HuIFN-β的表达。更多还原

【Abstract】 With the development of plant molecular biology and genetic engineering, plant-based expression system have emerged as a new promising force in the large-scale biopharmaceutical protein production for preventation or therapy. For some products, transgenic plants offer many potential advantages over traditional systems based on microbial or animal cells, or transgenic animals. A major advantage is the comparatively low cost of large-scale production. Plants also have the ability to process post-translational modifications, a low risk of contamination by organisms pathogenic, oncogenic DNA sequences, and endotoxins to humans and animal. Furthermore, plants or plant products which are edible provide the additional unique opportunity of serving as vehicles for oral delivery of the vaccine. However, several challenges remain to be met in terms of increasing yields, improving glycoprotein authenticity, removing processing bottlenecks and addressing biosafety and acceptability issues, as well as industry inertia. So finding new plant-expression host and exploiting new plant expression systems are the focus of plant-base expression study.Using Leaf lettuce(Lactuca sativa L.sp) as plant material, Agrobacterium vacuum infiltration transient expression system and tissue culture system were discussed and optimized. Employed the optimized transient expression system, expression vectors containing four IFN genes were transferred to lettuce leaves. Western blot and antivirus detection were employed to analyze these four genes expression and bioactivity. The main results were as follows:1. Established a high efficient transient expression system in lettuce. Intact lettuce leaves infiltrated with 200μM acetosyringone and 0.8 OD600 bacterial suspensions under vacuum for 30 minutes, then co-cultured at 24℃for 6 ds had the highest transient expression level. Used A1B1C1D1(0μmol/L acetosyringone, 0.4OD600 bacterial suspensions, vacuum for 10min, vacuum for 30 minutes 2d) as control,the optimized system produced a maximum GUS protein of 2.5% TSP with 21.39nmol?mg-1?min-1 MU activity, which was nineteen times of the control (1.31 nmol?mg-1?min-1 MU). The result indicated that the established transient expression system can significantly enhance transgenic gene expression.2. HuIFN-beta was successfully expressed as a 27kDaa glycoproein with plant special glycosylation in lettuce leaves. Antivirus bioactivity detection confirmed that the HuIFN-beta achieved by agrobacterium infiltration could inhibit VSV cytopathic effect in human amnionic (WISH) cells. By site-specific mutagenesis with serine (Ser) substitute cysteine (Cys) at the 17th in human interferon-beta gene, the influence of Cys17 on the normal bisulfur bond of Cys31 and Cys141 was elimating, and the space structure of interferon-beta became more stable. So the mutant interferon-beta had higher expression level. Antivirus bioactivities of four interferon-beta types (IFN,interferon-beta without signal peptide; sIFN, interferon-beta with signal peptide;mIFN,interferon-beta with mutant; smIFN, interferon- beta with signal peptide and mutant) were 3.1×104IU/mL, 5.8×104IU/mL, 6.3×104IU/ML, and 9.8×104IU/mL respectively. To our knowledge, it is the first detailed orthogonal optimizing study of Agrobacterium mediated transient expression and the first report on the production of the biologically active therapeutic proteins produced by Agrobacterium mediated transient expression in lettuce. In summary, transient expression by Agrobacterium vacuum infiltration can be adopted as an efficient, inexpensive and small-scaled plant expression system for therapeutic protein production.3. Constructed plant expression vector pBI121-F containing the gene of PNGase F with the aim to discuss glycosylation of interferon-beta in lettuce. Individual Agrobacterium cultures (GV3101) carrying the 35S: F and the 35S: IFN constructs were mixed together and infiltrated into leaves of lettuce. The result of Western blot indicated that: the molecular weight of IFN is not changed. This maybe because the expression level is too low to detect or moreα-1, 3-Fucose contained in plant glycosylation blocked the process of PNGase F.4. Cotyledons and leaves of three genotype leaf lettuce (Lactuca sativa L cultivars: Japanese Lettuce, American Grand Rapid, Thailand Lettuce) at 10-day were excised and cultured on MS basal medium supplemented with different combinations of hormones. A high efficient plant regeneration system adaptive to three different genotypes was developed in our study. MS-medium supplemented with 0.1-0.5 mg/L N6-benzylaminopurine (6-BA) in combination with 0.1mg/Lα-naphthaleneacetic acid (NAA) was the most effective to induce adventitious shoot directly. Genotype and explant type all have significant effects on shoot regeneration efficiency in terms of the percentage of explants producing shoots and the number of shoots produced per explant. Different genotype has its own optimal shoot-inducing medium and leaf is more responsive than cotyledons for shooting on the same culture conditions. Regenerated shoots were highly rooted on 1/2MS basal medium just supplemented with 0.1mg/L NAA alone. In addition, we also discovered that 50 mg/L kanamycin is enough for selection in gene transformation for different genotype leaf lettuces. These protocols will facilitate explants regeneration and gene transformation for a range of genotype lettuces.5. Used overlap extension PCR, constructed a fused gene with GUS and IFN with the aim to increase the expression level of IFN.更多还原