一种碱裂解菌液直接电泳快速筛选重组子的方法

目的：从碱裂解法提取质粒DNA的原理．经过实验摸索获得一种快速、经济、结果可靠的菌液直接碱裂解电泳筛选重组子的方法。方法：不需提取质粒DNA．只需将细菌培养液碱裂解后直接进行普通琼脂糖凝胶电泳分析,就可以快速筛选出转化重组子。结果：结果和提取质粒酶切鉴定鉴定结果一致。结论：经实验证明菌液直接碱裂解电泳筛选重组子是一种快速、经济、可靠的方法。     

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。     

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。     

一种经济高效的用Silica提取纯化质粒DNA的方法

目的：为了达到批量提取质粒DNA的目的,在多次实验的基础上,建立一种经济、高效的质粒提取方法。方法：以pUC18、pET28b、pCAMBIA1304等3种质粒为材料,分别采用silica法和碱裂解法提取质粒DNA,通过质粒DNA浓度的紫外分光光度法定量测定、电泳分析和HindⅢ酶切鉴定,对两种质粒提取方法的效果进行了比较与评价;对silica法进行了改进和优化,进行大批量重组子的提取和验证。结果：silica法和碱裂解法提取质粒DNA效果相当,都可进行后续实验,但silica法具有经济、高效、无毒的优势。结论：silica法是一种简单、经济、高效的质粒提取方法,可用于批量质粒DNA提取。     

快速小量提取质粒DNA的两种方法

碱裂解法是进行分子克隆实验中质粒DNA制备最为常用的方法。在实际实验操作过程中,对该实验方法进行了改进,改进的碱裂解法解除了所提质粒DNA中RNA污染及RNase的可能影响,快速提取法使阳性克隆的筛选更加迅速,快捷。两种方法的改进使实验更加高效、简单、实用。     

碱裂解法提取质粒DNA的研究

对质粒DNA及其结构、形态和功能进行了综述 ,介绍了碱裂解法提取质粒DNA的基本步骤 ,并对其提取过程中的注意事项进行了剖析 ,分析比较了不同提取方法的优点     

pcD-awte候选疟疾DNA疫苗制备及其质量相关性分析

本实验室构建的疟疾DNA疫苗经动物试验表明具有很好的免疫原性,为申请临床试验,进行了制备工艺的研究。本研究将含pcD-awte质粒的大肠杆菌DH5α在发酵罐中发酵培养,碱裂解法粗提质粒,再依次通过Sepharose 6FF分子筛层析、Plasmidselect 亲硫吸附层析和Source 30Q离子交换层析精制获得质粒纯品,并对纯品进行质量分析。结果每升培养液可获得质粒纯品43.9mg,质量符合Ferreira等推荐的药用标准。     

碱裂解法提取重组质粒DNA及PCR验证

目的:筛选获得高质量质粒,为进一步克隆、测序奠定基础.方法:采用常规的碱裂解法从大肠杆菌重组质粒中提取质粒DNA,核酸检测仪测定提取质粒DNA产量和纯度,并用前期DDRT-PCR时采用的引物进行PCR验证性实验,琼脂糖凝胶电泳检测,并对电泳图谱加以分析和鉴定.结果:利用常规的碱裂解法提取重组质粒,通过酚和氯仿的抽提,可以有效地去除蛋白质杂质,用含有Rnase抑制剂的无菌水溶解质粒提取效果最好,得到的质粒DNA无RNA污染,纯度比较高,OD260/OD280比值介于1.8～2.0之间,OD260/OD230比值大于2.0,经PCR反应验证后与预计相符.结论:通过该方法获得的重组质粒纯度和浓度都比较高,且PCR验证与预计相一致,可以满足后续分子生物学实验的要求.     

CTAB选择性沉淀和纯化质粒DNA工艺的建立

通过直接在大肠杆菌碱裂解上清中加入十六烷基三甲基溴化铵(CTAB), 优化CTAB与质粒DNA量的比例、质粒DNA选择性释放溶液的选择和TritonX-114的使用, 建立了简单、易行的大规模质粒DNA纯化工艺。纯化质粒DNA的质量检测结果显示, CTAB纯化的质粒DNA无菌体RNA污染, 菌体基因组DNA、内毒素和蛋白含量分别小于 100 ng/mg、50 EU/mg和10 mg/mg质粒DNA, OD260/OD280比值介于1.75~1.85之间, 超螺旋质粒DNA的比例大于80%, 该工艺纯化的质粒DNA能达到或接近FDA规定的人用质粒DNA的各项指标, 整个过程不使用动物源性酶和苯酚、氯仿、无水乙醇等有毒或易燃、易爆试剂, 成本低廉, 工艺环保。     

农杆菌质粒DNA提取方法的改良与鉴定

常规的农杆菌质粒DNA提取常采用碱裂解和酚、氯仿提纯方法,得率较低,一般在50ng/μl以下。针对常规碱裂解法的不足,利用含有pCGⅡ双价载体的LBA4404菌株通过增加菌液收集量、改变提取流程中的反应条件等对常规方法进行了改良,去除了酚、氯仿提纯过程,减少了提取过程对人体的伤害,得率一般在0.5-2μg/μl。所提取质粒DNA可直接用于PCR、限制性酶切等分子生物学试验。     

A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF

Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.     

Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia

The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24?±?0.41?g of pharmaceutical pUDK-HGF from 1.0?kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of >?95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.     

基因治疗用质粒DNA生产面临的问题及研究进展

基因治疗已成为21世纪一些重大疾病的有效治疗策略,目前携带治疗基因的重组质粒已作为基因药物进入临床研究。对用于基因治疗的生物制品的生产与质量控制都有相当严格的要求。虽然已建立大规模符合药学规格的质粒DNA生产工艺,能满足临床需求,但在这些生产工艺中还存在一些难以克服的瓶颈,如:载体构建、细胞裂解、细菌染色体DNA去除、细菌内毒素去除、生产过程中质量控制等。就近年来大规模生产临床用质粒DNA遇到的相关问题及解决方案作一综述。     

Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids

A new plasmid, pLS101, was constructed for use as a vector for cloning in Streptococcus pneumoniae. This plasmid carries two selectable genes, tet and malM, each of which contains two or more restriction sites for cloning. Insertional inactivation of the malM gene allowed direct selection of TcRMal- clones containing recombinant plasmids. Other means of enriching a recipient population for cells containing recombinant plasmids were examined. The effect of removing vector terminal phosphate in attempts to clone heterogeneous DNA fragments, such as those from chromosomal DNA, was to abolish recombinant plasmid establishment altogether, presumably because donor DNA processing during entry into the cell prevented establishment of the hemiligated molecule. However, with homogeneous DNA fragments, such as those from plasmid or viral DNA, vector phosphate removal allowed enrichment for recombinant plasmids. In the cloning of heterogeneous DNA that was homologous to the recipient chromosome (i.e. chromosomal DNA from S. pneumoniae), recovery of recombinant plasmids could be enriched tenfold (relative to the regenerated vector) by the process of chromosomal facilitation of plasmid establishment. This involved an additional passage of the mixed plasmids in which interaction with the chromosome of plasmids containing chromosomal DNA inserts (i.e. recombinant plasmids) increased their frequency of establishment relative to the vector plasmid. An overall strategy for cloning in S. pneumoniae, depending on the nature of the fragment to be cloned, is proposed.     

A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of genomic DNA from plasmid DNA by selective renaturation with immobilized metal affinity capture

In contrast to proteins, many nucleic acids can undergo reversible modification of their conformations, and this flexibility can be used to facilitate purification. Selective renaturation with capture is a novel method of removing contaminating genomic DNA from plasmid samples. Plasmid DNA quickly renatures after thermal denaturation and cooling (or alkaline denaturation followed by neutralization), whereas genomic DNA remains locally denatured after rapid cooling in mismatch-stabilizing high ionic strength buffer. Partially denatured genomic DNA can be selectively bound to a metal chelate affinity adsorbent through exposed purine bases, while double-stranded renatured plasmid DNA is not bound. Using this method we have readily achieved 1,000,000-fold clearance of 71 wt % contaminating E. coli genomic DNA from plasmid samples.     

An apparatus to control and automate the formation of continuous density gradients

Triton X-114 temperature transition extraction has been considered to be a simple and cost-effective strategy to eliminate endotoxin from plasmid preparations. However, a repeated cooling-heating process may promote the degradation of plasmid DNA. Based on the finding that the cloud point of Triton X-114 solution increases substantially in the presence of small amounts of sodium dodecyl sulfate (SDS) and that electrolytes decrease the cloud point of Triton X-114-SDS solution drastically, we designed a Triton X-114 isothermal extraction method for removing endotoxin from plasmid samples and found that it has the same endotoxin removal efficiency when compared with the temperature transition extraction method.     

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.