乳腺生物反应器的表达优化策略

乳腺生物反应器是指将外源基因导入动物基因组并在动物乳腺中特异性表达,利用动物乳腺合成、分泌蛋白的功能,在其乳汁中获得外源蛋白的技术。乳腺生物反应器凭借其高表达、低成本以及合成蛋白质的结构接近天然蛋白质等优势而被视为药用和营养蛋白生产的一次技术革新,然而由于外源基因随机整合以及重组蛋白表达不稳定等问题极大地限制了其应用。本文结合乳腺生物反应器的发展现状,从利用基因编辑技术、筛选合适的外源基因整合位点以及改进外源基因调控序列3个方面对乳腺生物反应器优化策略进行了综述,以期为提高乳腺生物反应器生产重组蛋白的表达提供理论借鉴。     

乳腺生物反应器的研究现状

乳腺生物反应器是将外源基因在哺乳动物的乳腺中特异表达,以转基因动物的乳腺组织生产药用蛋白。采用乳腺生物反尖器生产药用蛋白质是一种全新的生产模式,它已经成为生物技术领域发展的重要方面。乳腺生物反应器具有能够生产出具有完全生物活性的药用收白,纯化简单,投资少,成本低,对环境没有任何污染等优点,转基因动物生产药用蛋白可以获得巨额经济效益。     

利用转基因植物表达药用蛋白

随着药物生物技术和植物基因工程迅速发展 ,转基因植物被用作生物反应器生产具有医疗价值的多肽和蛋白质已成为生物医学研究的热点。研究表明转基因植物表达的蛋白质能够保持原有的结构和功能 ,这预示它将为药用蛋白的生产提供一条安全和廉价的新途径。主要概述了近年来国内外转基因植物生产诸如疫苗、抗体和其他药用蛋白或多肽等的研究进展 ,并着重探讨了存在的问题和解决策略。     

利用植物生物反应器生产药用蛋白的研发现状

<正>植物生物反应器具有成本低、安全性高等优点,且植物具有真核细胞表达体系,能进行准确的蛋白修饰,使产品的免疫原性及生物活性较高,因此,植物生物反应器应用日益广泛。文章就现阶段植物生物反应器生产药用蛋白的研发及应用现状进行了综述,分析了目前本领域存在的主要技术瓶颈问题,并对利用植物生物反应器生产药用蛋白的发展前景进行了展望。     

利用动物乳腺生物反应器生产药用蛋白

动物乳腺生物反应器是利用动物乳腺特异性启动子调控元件指导外源基因在乳腺中特异性表达,并从转基因动物奶液中获取重组蛋白。应用动物乳腺生物反应器生产药用蛋白具有生产方式简单,产量大,蛋白能进行翻译后修饰等优点,是具有广阔前景的生物医药产业。本文仅就动物乳腺生物反应器的建立、检测、目的蛋白的分离纯化以及存在的问题等作一综述。     

动物乳腺生物反应器的研究进展

利用转基因动物反应器生产药用蛋白是生物技术领域的一次革命,而通过动物乳腺是最好的渠道,药物蛋白可通过转基因动物的乳腺随乳汁分泌出来,产量高、易纯化,因此乳腺类似于一个制药工厂。国外已有多种蛋白通过乳朱反应器来制备,有些已进入临床研究阶段。本文主要介绍乳腺生物反应器的研究进展情况。     

叶绿体转基因植物--一种新型生物反应器

叶绿体转基因植物作为生物反应器,具有外源蛋白表达量高和环境安全性好等优点,近年来呈现出诱人的发展前景。本文综述了叶绿体基因工程的优越性,并重点介绍了叶绿体转基因植物作为生物反应器在生产疫苗、药用蛋白及生物可降解塑料等物质方面的最新研究进展。     

重组蛋白在植物体中的表达及其应用

近年来,以植物为生物反应器异源表达和生产具有药用及商业价值的蛋白质发展迅速,某些产品已进入临床试验,且获得了很好的疗效;另一类在植物体中表达的具有重要农艺价值的蛋白质可介导植物抵抗病原体,即植物抗体介导的抗性,是植物抗病原体分子育种的又一途径。介绍了重组蛋白在植物体中表达及其应用的现状。     

叶绿体转基因植物——一种新型生物反应器

叶绿体转基因植物作为生物反应器, 具有外源蛋白表达量高和环境安全性好等优点, 近年来呈现出诱人的发展前景。本文综述了叶绿体基因工程的优越性, 并重点介绍了叶绿体转基因植物作为生物反应器在生产疫苗、药用蛋白及生物可降解塑料等物质方面的最新研究进展。     

重组蛋白在植物体中的表达及其应用

近年来,以植物为生物反应器异源表达和生产具有药用及商业价值的蛋白质发展迅速,某些产品已进入临床试验,且获得了很好的疗效;另一类在植物体中表达的具有重要农艺价值的蛋白质可介导植物抵抗病原体,即植物抗体介导的抗性,是植物抗病原体分子育种的又一途径.介绍了重组蛋白在植物体中表达及其应用的现状.     

利用植物系统表达重组蛋白的的研究进展

植物是可以生产不同生物药剂品的成本低廉的生物反应器,综述了稳定转化系统、瞬时表达系统以及叶绿体基因组转化方法,这三种不同的植物表达系统的特点和研究现状,并对其存在的问题及未来的前景进行了分析。     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Methods for Baculovirus Amplification - Application to Large Scale Recombinant Protein Production in Insect Cells

Baculovirus amplification in one insect cell line Spodoptera frugiperda (Sf21) and subsequent recombinant protein production in another cell line, Trichoplusia ni, has been achieved within a single bioreactor. The advantages of this single bioreactor configuration include minimization of the virus volumes and titres required for large scale protein expression experiments as well as optimization of the infection process itself.     

转基因植物作为生物反应器生产口蹄疫疫苗的研究进展

利用转基因植物作为生物反应器表达重组蛋白,生产外源蛋白质作为动物疫苗是一个很有吸引力的廉价生产系统,它有可能代替生产成本较高的传统疫苗的发酵生产系统。通过口蹄疫病毒VP1结构蛋白基因在转基因植物中的表达,口蹄疫疫苗已在植物中产生。在植物中生产的抗原能够保持其自身的免疫原性。本文简要综述了近十年来用转基因植物作为生物反应器生产口蹄疫疫苗的研究进展、特点及其应用前景 。     

莱茵衣藻叶绿体生物反应器研究进展

莱茵衣藻(Chlamydomonas reinharditi)是一种遗传机制已研究比较清楚的模式植物。近年来,生物反应器是当今世界上各国生物技术研究的一个热点,随着生物技术的发展,已成功实现衣藻作为生物反应器生产重组蛋白及抗体,生产的部分产品已经实现了商品化,与其他生物反应器相比,其在外源基因表达水平和转基因植物安全性等方面有明显的优势,尤其是在控制转基因沉默和遗传稳定性方面展示了极大的优越性。因此,莱茵衣藻是一种具有很好发展前景的生物反应器,必将在未来的药用蛋白生物技术领域发挥重要作用。主要对提高基因在莱茵衣藻叶绿体中表达的策略,转化技术的特点及其未来的发展前景等方面进行了简单评述。     

Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth – associated production of a foreign protein, β-galactosidase

Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.     

Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures

Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post‐translational modification. In this study, bioreactor production of recombinant human alpha‐1‐antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25‐fold increase in extracellular functional rAAT (603 µg/L) and a higher ratio of functional rAAT to total rAAT (85–90%). Surprisingly, sustained rAAT production and steady state, long‐term bioreactor operation is possible following chemical induction and establishment of the viral amplicons. Biotechnol. Bioeng. 2010; 106: 408–421. © 2010 Wiley Periodicals, Inc.     

Bioreactor engineering for recombinant protein production in plant cell suspension cultures

A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications.     

Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng

The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.