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Expression, Identifying and Bioactivity Analysis of Human Interferon-β Produced in Lettuce

Author: LiJing
Tutor: ShenFaFu
School: Shandong Agricultural University
Course: Crop Genetics and Breeding
Keywords: Lettuce Agrobacterium mediated Interferon-beta GUS Vacuum infiltration Transient expression Tissue culture
CLC: S636.2
Type: PhD thesis
Year: 2007
Downloads: 219
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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.

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CLC: > Agricultural Sciences > Gardening > Vegetable gardening > Green leafy vegetables > Lettuce ( lettuce )
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