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به نا م خدا گياهان ترانسژنيك مينا ابراهيميان

به نا م خدا گياهان ترانسژنيك مينا ابراهيميان.

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به نا م خدا گياهان ترانسژنيك مينا ابراهيميان

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  1. به نا م خدا گياهان ترانسژنيك مينا ابراهيميان

  2. وارد كردن ژن‌هاي فراوان (مربوط به صفات مختلف) به ده‌ها گونه گياهي مانند گندم، جو، گوجه‌فرنگي، ذرت،سيب زميني، سويا، پنبه، مارچوبه، تنباكو و چغندرقندجهت اصلاح يا بهبود فرآورده‌هاي كشاورزي، امكان تغيير ژنتيكي در راه‌هاي بيوسنتزي گياهانبراي توليد انبوه موادي مانند روغن‌هاي خوراكي، موم‌ها،چربي‌ها و نشاسته‌ها كهدر شرايط عاديبه ميزان بسيار جزيي توليد مي‌شوندوكنترل افات زيستي تنها نمونه هاي كوچكي از كاربردهاي گياهان تراريخته هستند ه

  3. كاربرد پادتن در گياه 1-باعث تغيير فنوتيپ گياه ميشود(توليد دانه هاي با كيفيت بالا تر) 2-گياه نسبت به عوامل بيماريزا مقاوم ميشود 3-در راههاي متابوليسمي گياه تغيير ايجاد ميكند توليد فراورده هاي زيستي در گياهان نسبتا كم هزينه است زيرا از تجهيزات كافي براي ايجاد مواد گياهي برخوردار هستند اما به دليل اين كه توليد محصول بايد در محيط كنترل شده مانند شرايط استريل ازمايشگاه انجام شود جهت توليد پروتيين واجد ارزش تجاري استفاده نميشود .

  4. مراحل مهندسي ژنتيك براي توليد پادتن: 1-انتخاب ژن مورد نظر 2-جداسازي ژن مورد نظر 3-وارد كردن ژن مورد نظر در حامل 4-تكثير ژن در ميزبان مناسب 5-انتقال حامل ژن به سلول هدف 6-تكثير سلول هدف 7-توليد انبوه محصول ...

  5. سطح بيان ژن در گياهان مختلف است ميزان تجمع پادتن به نحوي آشكار به راههاي ترشحيهدف وابسته است. براي بيان يكسان پادتن بايد: 1- بيان متناسب زنجيره ها 2- زمان بيان انهايكسان باشد 3- سلولهاي هدف كه اين پادتن ها و آن مجتمع پيدا ميكنند يكسان باشد .

  6. پادتن هاي مونوكلونال از جمله مهمترين محصولات پروتيين دگر ساخت هستند كه در گياهان به منظور كاربرد هاي درماني يا تشخيصي توليد ميشود پادتن هاي توليد شده توسط گياهان از مرغوبيت بيشتري نسبت به پادتن هاي ميكروبي برخواردارند چون مراحل تغيير پس از ترجمه ورونويسي را بهتر انجام ميدهند براي مثال: بيان انتي بادي مونوكلونال v hh عليه انتي ژن muc1 در گياه توتون

  7. انتي بادي مونوكلونال تك دوميني vhh با منشا شتري به دليل دارا بودن ويژگي هاي: - تشابه بسيار بالا با زنجيره سنگين انتي بادي هاي انساني - حلاليت - تمايل واتصال اختصاصي به انتي ژن در سال2004 از منشا شتريك ودو كوهانه تهيه گرديد اين انتي بادي در تشخيص ودرمان بسياري از بيماريهاي سرطاني مورد توجه قرار گرفت .

  8. مراحل بيان انتي بادي تك دوميني vhh برعليه انتي ژنmuc1 در گياه توتون: -انتخاب ژن مورد نظروتحت كنترل قرار دادن ان با پيشبرنده camv35s وخاتمه دهندهnos -افزودن توالي افزايش دهنده به انتهاي 5 ژن براي بالا بردن بيان ان -انتقال ان به اگروباكتريوم(حامل) -انتقال ژن به وسيله حامل به ژنوم توتون(ميزبان) -اناليز گياه تراريخت با اليزا وpcr و وسترن بلات نشاندهنده بيان ژنvhhدر گياه تراريخت توتون ميباشد ميزان بيان1/12-1/63درصد از كل پرتيين هاي محلول را تشكيل ميدهد .

  9. Anti-IgE Antibody Production in Transgenic Corn ~ Process Design and Practical Consideration Chien-Chung Chen, Tzu-Rung Shiu and Hisao-Ming WanDepartment of Chemical Engineering, National TsingHua UniversitySummaryExpression in transgenic plants is potentially one of the most economicalsystems for large scale production of valuable protein products. We present acomplete study of anti-IgE antibody production in transgenic corn from upstreamproduction to downstream purification. In genetic construction, all procedures aredescribed step by step. In purification, we have designed a realistic process to produce200 kg of anti-IgE antibody annually. The process is similar to other systems exceptfor initial corn milling and protein extraction. The relative low recovery is come fromprotein lost during milling. Furthermore, some key issues, including production,security, environmental safety and economic analysis are also discussed. We concludethat corn-produced antibody is promising, but one should put strong emphasis onsocial issues and ethical issues.Introduction1~5Anti-IgE antibody is a completely different type of asthma medication. It worksby interrupting the cascade of asthma or allergic events by inhibition IgE before itbinds to the mast cell. Anti-IgE antibody manufactured from mammalian culture hasbeen approved by FDA for use in the treatment of asthma patients. (Xolair® , June 20,2003). Improved asthma drugs are urgently needed. The number of people withasthma in the USA has grown from 6.7 million in 1980 to more than 17 million today.About 60% have allergic asthma. Each year, asthma sufferers account for 500,000hospital admissions and 2 million emergency room visits. Asthma costs the economyan estimated $11 billion every year. Analysts estimate that the annual market foranti-IgE antibody is about $1 billion, with the cost around $10,000 per patient.Many of the antibodies currently produced in plant-based expression systems arehigh-value products for pharmaceutical use. Indeed, plants represent cost-effectivesystems for the large-scale production of pharmaceuticals and freedom fromanimal-derived pathogens (prions and viruses). Moreover the corn production systemprovides additional easy storing and shipping properties that allow high flexibility inbioprocess management.

  10. The production process is illustrated with figure 1. Transgenic corn seed can be milled and then extracted. The resulting extract is clarified before chromatographic separation steps begin. Final purification consists of protein A affinity column and two ion-exchange column. The process yield is around 60% from corn seed to drug substance. Genetic Construction5~6 Construction of Plasmid Used for Transformation The amino acid sequence of humanized anti-IgE antibody cDNA was reverse translated into nucleic acid sequence utilizing a preferred corn codon usage table. From this computer-generated synthetic sequence, overlapping, complementary oligonucleotides with compatible restriction site termini were designed, then annealed and ligated to yield the corn optimized gene. A C-terminal Lys-Asp-Glu-Leu (KDEL amino acid sequence for ER retention was also synthesized with corn-preferred codon.Compatible restriction sites between these two gene fragments were ligated, with theER retention sequence at the 3’ end of the anti-IgE gene. The linked segment wascloned into a vector derived from pBlueScript SK+ as a backbone. In this plasmid, theER retention signal sequence/anti-IgE gene fragment was inserted between the maizeubiquitin 5’ region, which includes the promoter, the first exon and first intron and thepotato proteinase inhibitor II (PinII) transcription terminator region. The resultantplasmid is pAnti-IgE (Figure 2A). The expression cassette for herbicide resistance isplasmid pBAR, which is composed of the tandem CaMV promoter followed by theAdH1 intron from corn, the bar gene and the PinII terminator. (Figure 2B)Transformation, Tissue Culture and Generation of Master Seed Bank (MSB)Particle bombardment was used to simultaneously introduce two plasmids intoan established callus line. Transformants expressing the bar gene were selected in thepresence of bialaphos (3 mg/l). Cotransformants that also expressed the anti-IgEgenewere identified by ELISA screening of the selected colonies. Multiple plants (T0generation) were regenerated from anti-IgEexpressing colonies, transferred to thegreenhouse and assayed for anti-IgEexpression in leaf tissue by PCR. T1 seed wasobtained by outcrossing, with the T0 plants as the female parent and an untransformedinbred line as the male parent. Using selection and a backcross program with elitegermplasm that maximizes agronomic characteristics, seeds with the highest anti-IgEexpression were obtained and stored as master seed bank (MSB).

  11. Manufacturing Process5~9 Process Description The process is divided into three sections (Figure 3). The front end of the process is a milling section where corn flour is produced. The second section consists of the aqueous extraction of protein from the corn flour, the removal of spent solids, concentration of the crude extract, and drying of the spent solids. The purification section includes three-stage chromatography (one protein A affinity column and two ion-exchange column) and diafiltration. The corn flour is produced by mill. The flour is produced only as needed, so no provision for storage is necessary. Two extraction tanks are used to allow continuous operation of the rotary vacuum filter and the spent solids dryer. The resultant crude extract is concentrated by ultrafiltration. The concentrated crude extract is exchange with buffer solution by diafiltration before loaded onto a protein A column to capture anti-IgE antibody. The bulk of the contaminant proteins are removed. The elution is exchanged with buffer. The purification proceeds using an anion-exchange (DEAE) column to remove DNA residual and further purification. Following is a diafilter before cation-exchange column (CM). CM column provide additional removal of impurities, leached protein A ligand and serves as a concentration step before the final formulation diafilter. Process Simulation and Economic Analysis The production plant will process 1444 kg of seed per batch containing 0.075% anti-IgE antibody. The total batch time was estimated to be about 54 h (Figure 4). The plant was planned to work 2 shifts a day, 7 days a week, and 44 weeks per year. Eight weeks are free for maintenance, holidays, personnel training sessions and dealing with the technical problems that might lead to production failure. The plant could process 308 batches producing 200 kg of anti-IgEIgE annually. A final purification yield ofabout 60% and purity of 99% is achieved. The column size is calculated by assuminglinear velocity and resin dynamic capacity and other equipment size and operatingcondition were modified slightly from those described by Roque L. al. (Table 1.)In economic analysis, Total operation and capital cost were estimated byadjustment factor described by Dominique M al. The resultant is $15.6 /g protein oftotal operating cost and $12 million of total capital cost.Remaining Considerations3~5, 10~11Transgenic corn should not be grown within a half-mile of any other corn, that itis not grown within a mile of any seed corn production, and that it is planted threeweeks before or after other corn crops within one mile.Security measures may include installing motion detectors or fences, postingguards or using other techniques to prevent animal intervention. For example, “birdwatchers” may sound horns to keep birds from entering a field during a criticalproduction stage.

  12. Contract growers are trained on APHIS procedures specific to Plant-made pharmaceutical production. SOPs for handling and shipping should be developed to prevent the unintentional contamination. Waste disposal is a serious problem in transgenic plant. The process described above extracting 200 kg anti-IgE antibody from 440,000 kg of corn produces 439,800 kg of byproduct that contains another 133 kg of anti-IgE antibody. The byproduct is incinerated because anti-IgE antibody is not proved its safety for long-term and high-dosage use. Conclusions Corn-based production system shows its industrialization and commercialization potential of manufacturing antibodies, especially for those in great demand. In this study we have design a technically feasible process to produce 200 kg anti-IgE antibody annually. On the basis of our economic studies, comparison to other expression system, production cost of anti-IgE antibody form transgenic corn is markedly the most cost-effective. But there are also many remaining challenges in this area, including incomplete glycosylation, regulatory issues, waste disposal problem and maybe the most important for commercial production, social issues and ethical issues. From technical, engineering and economic viewpoints, corn-based expression system is promising. Once a corn-produced MAb is marketed, the severely limited manufacturing capacity today can be resolved. References 1. Hamelmann E, et al. “From IgE to Anti-IgE: Where do we stand?” Allergy. 57 (11),983-994. (2002)2. http://www.gene.com/gene/3. Stoger E, et al. “Plantibodies: applications, advantages and bottlenecks” CurrOpinBiotechnol. 13(2), 61-66. (2002)4. Hood EE, et al. “Monoclonal antibody manufacturing in transgenic plants--myths and realities” CurrOpinBiotechnol. 13 (6), 630-635. (2002)5. Baez J, et al. “Corn seed production of therapeutic proteins moves forward - One company'sexperience” Biopharm. 13 (8), 50-54. (2000)6. Hood EE, et al. “Commercial production of avidin from transgenic maize: characterization oftransformant, production, processing, extraction and purification” Molecular Breeding 3: 291–306.(1997)7. Mison D, et al. “The industrial production costs of recombinant therapeutic proteins expressed intransgenic corn” Biopharm. 13 (5), 48-54. (2000)8. Roque L, et al. “Process and Economic Evaluation of the Extraction and Purification ofRecombinant ‚β-Glucuronidase from Transgenic Corn” Biotechnol. Prog. 14, 607-614. (1998)

  13. 9. Hsrish L, et al. “Considerations during development of a protein A-based antibody purification process” Biopharm. 15 (1), 14-20. (2002) 10. Crosby L. “Commercial production of transgenic crops genetically engineered to produce pharmaceuticals - Agriculture technology already exists to address most GMP issues” 16 (4), 60-67. (2003) 11. http://www.aphis.usda.gov/ppq/biotech/pdf/pharm-2002.pdf pAnti-IgEpBAR Figure 1. Production scheme Harvest 1.5 kg crude MAb/acre Dry and clean before storage Stored at 5 °C Corn seed Stored corn see Aqueous extractionClarifyChromatography (3 steps)≈ 60% recovery≈ 0.9 kg pure MAb BulkactiveFeedstockMSB generationWSB generationProduction fields (2 crops/yr)Stable reservesFigure 2. Restriction endonuclease maps of plasmids used for corn transformation to generate the anti-IgEAntibody line. A. pAnti-IgE is composed of the corn ubiquitin promoter and nontranslated first exon withintron, a gene encoding humanized anti-IgE Antibody, a ER retention sequence and the PinII terminatorcloned into a Bluescript SK+ plasmid backbone. B. pBAR is composed of the tandem CaMV promoterfollowed by the AdH1 intron from corn, the bar gene and the PinII terminator cloned into Bluescript SK+.

  14. مجله بيوتكنولوژي دانشگاه تربيت مدرس 1.Abdel-Wahab Z, Weltz C, Hester D, Pickett N, Vervaert C, Barber JR, Jolly D, Seigler HF (1997) A Phase I clinical trial of immunotherapy with interferon- gene-modified autologous melanoma cells: monitoring the humoral immune response. Cancer, 80: 401-12. 2.Azhdari H (2009) Cloning and transformation of human IFN gene to tobacco plants. M.Sc. thesis. Department of Biotechnology, Faculty of Agriculture, TarbiatModarres University. Tehran - Iran. 3. Bagheri KH (2009). Gamma-Oleosin interferon gene transfer to Canola and study of transgenic plants. Ph.D. Thesis. Department of Plant Breeding, Faculty of Agriculture TarbiatModarres University. Tehran - Iran. 4. Boothe JG, Parmenter DL, Saponja JA (1997) "Molecular farming in plants: Oilseeds as vehicles for the production of pharmaceutical proteins. Drug Development Research, 42(3-4): 172-181. 5. Borisjuk NV, Borisjuk LG, Logendra S, Petersen F, Gleba Y, Raskin I (1999) Production length=0.25m

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