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。     

质粒pcDNA3—HGF的大规模纯经制备研究

质粒ｐｃＤＮＡ３－ＨＧＦ具有潜在的临床治疗缺血性疾病的应用前景。大规模纯化制备是质粒ＤＮＡ应用于基因治疗的关键步骤。质粒大规模纯化制备流程包括：发酵、离心收集细胞、碱性裂解、Ｑ－Ｓｅｐｈａｒｏｓｅ ＸＬ捕获质粒ＤＮＡ、Ｓｏｕｒｃｅ １５Ｑ精制质粒ＤＮＡ,所得纯超螺旋质粒ｐｃＤＮＡ３－ＨＧＦ符合质量标准。该纯化制备方法避免使用动物源性的酶及有毒试剂。     

重组人促红细胞生成素中试纯化工艺研究

采用堆积床生物反应器,用无血清培养基培养分泌ｒｈＥＰＯ的工程细胞株ＸＰ９５０１。所收集的上清液,经过快速离子交换层析—反相—分子筛层析纯化后,所得ＥＰＯ纯度达９９％以上,比活性为１－５×１０５ＩＵ／ｍｇ。整个纯化全过程的ＥＰＯ体内活性回收率为４６％。所纯化的ＥＰＯ分子量为３６ｋｄ,等电点为３－５。免疫印迹证明其有天然ＥＰＯ的免疫原性,Ｎ端１５个氨基酸序列分析与文献报道一致。本纯化工艺路线简单,时程短,重复性好,适合于大规模生产重组人促红细胞生成素。     

携带人肝细胞生长因子重组质粒pUDK-HGF对大鼠肾纤维化的防治作用

目的:研究肝细胞生长因子(HGF)基因转导对庆大霉素诱导的大鼠肾纤维化损伤的防治效果。方法:以雄性Wistar大鼠腹腔注射硫酸庆大霉素注射液制备肾纤维化模型;实验分为正常对照组、肾纤维化模型组、HGF治疗组;造模后第30 d,HGF治疗组于左侧肾脏直接注射重组质粒pUDK-HGF注射液,模型组注射质粒pUDK,正常对照组只进行假手术;于造模后第60 d处死大鼠,评价HGF对血尿素氮、血肌酐、24 h尿蛋白、肾系数等肾功能指标的改善作用,并对肾纤维化进行组织学评价。结果:与正常对照组相比,模型组肾功能下降,肾系数(8.8±0.95 g/kg)、血尿素氮(9.4±2.61 mmol/L)、血肌酐(42±10.33μmol/L,P<0.05)及24 h尿蛋白定量(25.78±8.66 mg,P<0.05)升高;HGF治疗组对肾功能有所改善,可缓解肾纤维化的进展。此外,本实验表明,对已纤维化肾脏直接注射HGF基因,可促进肾间质血管再生,并进一步降低肾小管间质损伤积分。结论:将HGF基因靶向导入大鼠体内可有效防治肾纤维化。     

重组质粒pUDKH的质量分析

目的:分析研究pUDKH的质量。pUDKH是由本实验室构建的携带人肝细胞生长因子(HGF)基因的真核表达质粒,具有治疗肢体动脉闭塞病的应用潜能。方法:用光密度法分析pUDKH的浓度及纯度;用0.8%琼脂糖凝胶电泳分析超螺旋pUDKH比例及RNA;以地高辛标记的核酸探针固相斑点杂交法测定残留宿主DNA含量;用酶联免疫法测定残留宿主菌蛋白;以4组限制性内切酶分析一致性;PCR扩增目的基因片段;抑菌圈测定板测定残余抗生素;用鲎试剂检测细菌内毒素;对CHO细胞进行基因转染;ELISA检测上清HGF表达量;用Transwells法分析表达的上清液诱导ECV304细胞迁移数。结果:03批次pUDKH的浓度为1.97mg/mL,D260nm/D280nm值为1.84,超螺旋pUDKH比例为95.5%;电泳图谱中未见RNA;残留宿主DNA含量低于质粒DNA的0.2%;菌体蛋白质残留含量低于质粒DNA的0.1%;限制性酶切图谱与自行制备的对照品一致;PCR扩增到HGF基因片段,长约2.2kb;未见残余抗生素(卡那霉素)抑菌圈;细菌内毒素≤0.03125EU/mL;转染的CHO细胞上清HGF表达量为39.32ng/mL;表达的上清诱导ECV304细胞迁移数高于对照组2倍。结论:03批次pUDKH的质量符合药学规格。     

rhHGFα重组质粒的构建及其在大肠杆菌中的表达

目的 :建立人肝细胞生长因子α链 (rhHGFα)高效原核表达体系 ,分离纯化rhHGFα蛋白 ,检测其生物活性。方法 :以pRC CMV hHGF为模板 ,扩增hHGFαcDNA继以XhoⅠ切为两个片段 ,亚克隆入pBSKS,测序后 ,将上述两个片段与pBV2 2 0连接 ,构建重组质粒。转化大肠杆菌 ,筛选高表达菌株。分离包涵体 ,8mol L尿素溶解 ,稀释复性后纯化重组蛋白 ,MTT法检测活性。结果 :测序证实所克隆的hHGFα基因序列正确 ,筛选出高效表达菌株E .coliBY ,SDS PAGE显示在相对分子量 5 2 0 0 0处出现特异的目的蛋白表达带 ,表达量约占全菌蛋白的 2 5 %。表达产物以不溶性的包涵体 (IB)形式存在 ,复性后具有刺激原代培养成年大鼠肝细胞生长的作用。结论 :以大肠杆菌为宿主 ,成功表达了hHGFα蛋白 ,为进行hHGFα结构与功能研究及中试生产奠定了基础。     

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。     

一种高纯度、高得率的质粒DNA纯化法

一种高纯度、高得率的质粒ＤＮＡ纯化法汤乃梅,王晓民,韩济生（北京医科大学神经科学研究中心,北京１０００８３）关键词质粒ＤＮＡ纯化本文介绍一种方法,它不仅可以得到高纯度质粒ＤＮＡ,而且在同等纯度下的得率也高于其它方法,其操作流程如下：１．５００ｍｌ菌液...     

快速鉴定重组质粒菌落的新方法

快速鉴定重组质粒菌落的新方法董北阎锡蕴（康奈尔大学医学院医学系,纽约）（中国科学院微生物研究所,北京１０００８０）筛选含重组质粒菌落常用的方法有探针杂交、小规模制备质粒ＤＮＡ进行限制酶切鉴定、。互补及插人失活。我们在此介绍一种新方法,即半菌落ＰＣＲ扩...     

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.     

Kinetics of loss of a recombinant plasmid in Bacillus subtilis

Recombinant plasmid pCEDS is structurally unstable in Bacillus subtilis cultures. We have previously shown that stability can be independently increased by changing from a complex medium supporting high growth rates to a chemically-defined medium supporting a lower growth rate and removal of a 4.77-kb EcoRI fragment from pCED3 to give plasmid YS1. Further stabilization was achieved by combining the two approaches. In the present work, we show that the stabilization of the plasmid-encoded LacZ(+) phenotype can be explained solely by the effect on the growth rate ratio between cells containing modified and parental plasmids. By using modified stability experiments (where a single cell rather than a suspended colony was used to initiate growth), independent growth rate measurements, and a simple mathematical model, we can describe the kinetics of the loss of the LacZ(+) phenotype in terms of two variables, alpha and p (where alpha is the ratio of growth rates between modified and parental cells, and p is the probability of obtaining modified cells from parental cells). Under the conditions tested, the average values of alpha were 1.52 for cultures growing in complex medium, 1.28 for cultures growing in defined medium, and 1.18 for cultures containing the modified plasmid pYS1 growing in complex medium. The calculated p values ranged between 10(-8) and 10(-10) under all conditions. Plasmid (pYS137) was used to directly estimate plasmid deletion rates in B. subtilis and it showed a rate between 5 x 10(-8) and 1.1 x 10(-9) deletions/cell/generation. In contrast to B. subtilis, there were no detectable differences in growth rates between Escherichia coli strains harboring plasmid pCEDS and plasmid-free cells. These results explain the observed stability of pCEDS in E. coli cultures and indicate that readily detected instability in B. subtilis cultures can be the result of rare deletion events.     

人肝细胞生长因子基因表达质粒的构建及其活性研究

目的 :构建一种携带人肝细胞生长因子 (HGF)基因的表达质粒 (pUDKH) ,并对其体外活性进行研究 ,为其体内应用提供依据。方法 :从人胎盘cDNA文库用PCR方法克隆人HGF基因 ,并将其克隆至自行构建的真核表达载体 pUDK上 ,获得表达质粒 pUDKH。将pUDKH体外转染原代培养的骨骼肌细胞 ,分析其转染效率及表达上清中HGF、血管内皮生长因子 (VEGF)的表达水平 ,并采用MTT法分析不同剂量HGF表达产物对人脐静脉内皮细胞的作用。结果 :所克隆构建的携带人HGF基因的质粒 pUDKH可有效转染原代培养的骨骼肌细胞 (0 .0 5 7% ) ,并表达HGF(16～ 18ng/ 4× 10 5cells)和VEGF ,其表达产物对人脐静脉内皮细胞具有明显的增殖刺激活性 (P <0 .0 5 ) ,而且有剂量效应关系。结论 :初步证实本研究构建的质粒 pUDKH具有体内治疗缺血性疾病的应用潜力     

质粒治疗肢体动脉闭塞性疾病的安全性分析

肢体动脉血管闭塞性病是一种严重危害人类健康的疾病,许多病人最终要截肢,基因治疗为这类患带来了希望。利用肝细胞生长因子促进血管生成的作用,采用质粒作为载体对大鼠和故事片诉下肢动脉闭塞疾病模型进行治疗,效果良好。为探讨基因治疗的安全性问题,用绿色荧光蛋白报告基因研究质粒在体内的分布和表达,方法简便。通过制作冰冻切片观察绿色荧光蛋白在小鼠体内的表达情况发现局部注射质粒不会造成全身性扩散,表明该方法安全。     

重组人凝血因子Ⅷ表达质粒的构建及其在HepG2细胞中的表达

目的:构建含有B区缺失型(△760aa-1639aa)人凝血因子Ⅷ(B domain-deleted human FⅧ,BDDhFⅧ)的真核表达质粒,转染HepG2细胞使其稳定表达人凝血因子Ⅷ。方法:将BDDhFVIII基因片段插入pcDNA4/v5-his空载体中构建重组真核表达质粒,测序正确后电转入HepG2细胞,经Ni-NTA纯化,利用Western blot检测凝血因子Ⅷ在HepG2细胞中的表达,持续培养获得稳定表达BDDhFⅧ蛋白的细胞株。结果:经限制性酶切和测序鉴定均证实重组真核表达质粒pcDNA4/v5-his-BDDhFⅧ成功构建,在转染HepG2细胞后,Western blot检测证实人凝血因子Ⅷ可以在HepG2细胞中正确表达。结论:成功构建了人凝血因子Ⅷ的稳定细胞株,并能在HepG2细胞表达目的蛋白。     

Non-GMP plasmid production for transient transfection in bioreactors

We describe a generic plasmid purification process for producing DNA for larger-scale transient transfection. Data on plasmid quality with regard to residual protein, endotoxin content and presence of different plasmid forms is given. The effects of contaminants and plasmid forms on expression levels of TNFRp55 and SEAP are discussed. Transient transfection of serum-free suspension grown mammalian cells represents a suitable approach to provide research quantities of proteins (50–100 mg) within1–2 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.