大规模生产制备质粒DNA技术进展

质粒DNA疫苗或基因治疗剂是生物制品的新品种。鉴于质粒DNA表达效率低、持续时间短,需建立一套大规模生产制备质粒DNA的工艺,即发酵、碱性裂解、分离纯化及质量控制。目前质粒DNA大规模生产已经达到千克级水平,一些产品正在进行临床实验。     

临床用DNA疫苗生产工艺研究进展

DNA疫苗是继传统疫苗和基因工程蛋白亚单位疫苗之后的新一代疫苗,具有很好的市场潜力。DNA疫苗生产工艺的质粒DNA大规模制备技术对DNA疫苗的产业化具有重要意义。本文介绍了DNA疫苗生产工艺的研究进展包括提高质粒DNA产量的策略、发酵后处理工艺、有效去除杂质和分离超螺旋构象质粒DNA的纯化工艺以及DNA疫苗生产相关的质量控制体系等。     

微环DNA研究进展

质粒载体在基因治疗中占据重要地位.传统质粒DNA在真核生物中可能会引起严重的炎症反应,未甲基化的CpG序列可能抑制基因的表达,最好的解决办法是在生产质粒载体过程中将细菌调控序列整体消除.微环DNA是一种新颖的小环超螺旋表达框,它是传统质粒在大肠杆菌体内通过位点特异性重组得到的.微环DNA缺乏抗性标记基因、复制原点等细菌...     

质粒载体的基因治疗

质粒载体的基因治疗作为一类极有前途的非病毒基因治疗方法,具有低免疫反应,安全稳定,低成本,适合商业化生产等特点,本文综合了用于基因治疗的质粒载体的转染机制,转染方法,构建和大规模制备。     

胁迫诱导抗性基因转移导致细菌耐药的分子机制研究进展

抗性基因转移是细菌形成耐药性的重要原因.近年来的研究表明胁迫因子可通过多种机制诱导抗性基因转移.DNA损伤可导致细菌产生SOS应激反应,进而诱导接合DNA介导的抗性基因转移.在一些缺乏SOS系统的细菌中,抗生素胁迫可诱导细菌建立自然转化感受态.此外,作者最近的研究表明普通胁迫应答因子RpoS调控一种由双链质粒DNA介导的固体基质表面的抗性基因转移方式.本文在总结SOS依赖和非依赖型胁迫因子诱导细菌接合和转化介导的DNA转移以及RpoS调控固体基质表面双链质粒DNA转移的基础上,提出今后需重点研究胁迫因子如何激活关键调控蛋白以及这些调控蛋白如何影响DNA转移相关基因表达等关键问题.解决上述问题将为寻找合适的分子靶标用于防控抗性基因转移导致的细菌耐药奠定基础.     

线形质粒

质粒是染色体外的遗传因子,是一种双链环状的DNA分子,质粒本身含有复制的遗传结构,能在细胞质中独立自主地进行自我复制。它是一个复制子。一些质粒也能整合到细菌染色体中随染色体的复制而复制。质粒一般都带有某些抗性基因。     

应用菌落裂解法快速检测质粒DNA

某些细菌除了含有染色体DNA外,还带有质粒DNA。质粒DNA往往带有抗药性基因,并且与某些细菌病原性有关。另外它在遗传工程研究中是一种十分有用的运载体。因此无论在遗传工程技术,还是临床检验,都需要有一种方法将质粒DNA检出,确定其大小,作进一步的研究和分析。对于快速简便检测质粒DNA的方法已有一些报道。我们直接从琼脂平板上挑取菌落,并使其裂解,然后进行琼脂糖凝胶电泳,检测的效果是满意的。     

基因工程菌

生物工程如果从1972年美国斯坦福大学生物化学家P.伯格首次构成第一批重组DNA分子开始,迄今才18年的历史。在这短短的十几年中,由于生物工程学家的努力,生命科学迅速进入了人工改造和创造新生命的时代。生物工程技术的发展,是与某些细菌质粒工具的利用分不开的。由于质粒DNA分子只有细菌核物质DNA分子的1％,易于提取分离和纯化,又极易与目的基因重组,就成了基因工程生产不可缺少的手段。细菌体内的基因,如抗性基因、合成特殊产品的基因等又是人们培育抗逆良种、获取重要生化产品的目的基因。我们可以把运用于生物工程生产,为人类作出巨大贡献的细菌、真菌、放线菌称之为基因工程菌。     

整合子与细菌耐药性的相关研究进展

整合子是广泛存在于细菌中的一种可移动基因元件,它可以捕获外来基因盒并使其在细菌体内得到表达,在细菌耐药性的传播过程中扮演着重要角色。过去研究认为,细菌耐药是在质粒及转座子~([1])等基因水平上广泛传播,近几年大量研究表明,细菌可通过位点特异性重组的方式将耐药基因盒捕获并整合到自身的染色体或者质粒DNA上,即细菌体内存在一种天然的基因克隆表达系统——整合子。细菌耐药的高频次出现已成为临床医疗工作中的瓶颈,整合子不仅在细菌耐药中起关键作用,而且在细菌适应性及基因进化中具有普遍又重要的意义。     

遗传学及对生命的认识(续1)

1 克隆与拷贝DNA 在70年代早期,旧金山加利福尼亚大学的Her-bert Boyer和斯坦福大学的Stanley Cohen已经能够将取自别的细菌,尔后是无关的动植物细胞的基因插入细菌之上,使其克隆化。他们首先使用限制酶将供体DNA切割成可操作的片段。其次,他们发现如何将这些基因拼接入质粒,然后再由质粒将该DNA片段带入细菌。一旦进入细菌,新基因便随着细胞分裂而复     

重组质粒pUDK-HGF 的中试纯化工艺

pUDK-HGF是携带人肝细胞生长因子的裸质粒,目前已进入I期临床试验,因此需要大量符合药学规格的质粒DNA。文中建立了pUDK-HGF中试规模纯化制备的新工艺。流程包括：发酵、离心收获菌体、碱裂解、超滤浓缩碱裂解液、Sephacryl S-1000层析除去RNA并更换缓冲液、plasmidselect捕获超螺旋质粒DNA、琼脂糖凝胶6BFF除盐。新工艺可获得浓度为2.0 mg/mL、纯度在1.70以上的裸质粒原液,符合相关质量标准,并避免使用动物源性的酶及有毒试剂。     

Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Owing to the commercial interest in these approaches, the development of production and purification strategies for gene-therapy vectors has been performed in pharmaceutical companies within a confidential environment. Consequently, the information on large-scale plasmid purification is scarce and usually not available to the scientific community. This article reviews downstream operations for the large-scale purification of plasmid DNA, describing their principles and the strategy used to attain a final product that meets specifications.     

Animal-free production of ccc-supercoiled plasmids for research and clinical applications

The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)-an important tool for quality control and stability assessments in DNA storage or application. Hence, a large-scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal-derived substances. Using CGE, the development and in-line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms.     

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.     

重组大肠杆菌碱裂解方法的改进

为了降低质粒DNA的生产成本,对经典碱裂解法中的溶液III进行了改进,以表达溶菌酶基因的pcDNKLYZ重组质粒转化的大肠杆菌DH5α为指示菌,用标准碱裂解和改进碱裂解法提取质粒pcDNKLYZ,以提取的质粒产量和质量为指标,判断优化碱性裂解法的性价比,结果显示,用改进后的碱裂解法裂解重组菌,提取的pcDNKLYZ质粒产量和质量等指标与标准方法接近,而成本仅为标准方法的1/4,可用于重组质粒的大规模制备。     

Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.     

重组质粒pVAX1-PENK大规模制备研究

目的：建立质粒pVAX1-PENK的大规模制备2--艺。方法：对大肠杆菌工程菌DH5α-pVAX1-PENK进行补料发酵,利用自行发明的连续碱裂解过程对菌体进行裂解,经超滤浓缩后,用Sepharnse 6 Fast Flow层析柱分离DNA与RNA,再经Plasmidselect Xtra层析柱分离超螺旋质粒DNA与开环或线性质粒DNA,最后经Source 15Q层析柱精制质粒DNA。结果：发酵获得质粒pVAX1-PENK的产率为182mg／L,经碱裂解及层析分离后,最终制备的质粒DNA超螺旋比例大于98％,总回收率为60.5％,纯度（D260nm／D280nm）为1.8～2.0。结论：建立的质粒DNA生产工艺可以制备大量高纯度的质粒DNA,并避免了使用动物源性的酶及有毒试剂。     

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process

Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.     

Plasmid DNA production for pharmaceutical use: role of specific growth rate and impact on process design

Application of plasmid DNA as pharmaceutical to be used in gene therapy and vaccination has been investigated intensively in recent years. To be able to provide sufficient material that is in accordance with quality of pharmaceutical grade it is mandatory to gain comprehensive process knowledge which is even requested by regulatory agencies. Regarding plasmid DNA production the specific growth rate has been identified as one of the key parameters. The reduction of specific growth rate results in an increase of plasmid DNA formation. However, quantitative explanations that allow for efficient process development and design are still missing. The presented study proposes a model that clearly demonstrates the relationship between specific growth rate and plasmid formation due to identification of the specific plasmid production rate as relevant key parameter. In addition the model is proved to serve as a useful tool in process development and design.     

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi