聚(甲基丙烯酸-二乙烯基苯-三烯丙基异三聚氰酸酯)整体柱应用于质粒分析

针对质粒的结构特点,合成了含大量10~300 mm超大孔的阴离子型聚(甲基丙烯酸-二乙烯基苯-三烯丙基异三聚氰酸酯)(PGDT)整体柱,经二乙胺修饰的PGDT具有密度为1.58 mmol/g的阴离子交换功能基团. 将其用于质粒标准品和细胞裂解液的分析,并与市售的无孔微球型色谱柱TSK DNA-NPR和整体柱型色谱柱CIM DEAE Disk Monolithic Column比较. PGDT整体柱分析质粒标准品时能有效将质粒与其他杂质分离,效果与市售TSK和CIM色谱柱相当;分析细胞裂解液时,PGDT整体柱和CIM色谱柱能将RNA、非核酸杂质分离,TSK则仅能将质粒和非质粒分离. 合成的PGDT整体柱对质粒样品有良好的分析能力.     

Total DNA of Glycyrrhiza uralensis transformed into Hansenula anomala by ion implantation:Preparing Glycyrrhizic acid in recombined yeasts

<正>Glycyrrhizic acid(GA) in Glycyrrhiza uralensis(G.uralensis) is physiologically active.In this study,the total DNA of wild G.uralensis was randomly transformed into Hansenula anomala by implantation of low-energy Ar~+ and N~+,to produce five recombinant yeast strains relating to biological synthesis of the GA or Glycyrrhetinic acid (GAs).After culturing in liquid medium for 96 h,the resultant GA,18α-GAs and 18β-Gas were determined by reversed-phase high performance liquid chromatography(RP-HPLC),and the corresponding concentrations were 114.49,0.56,and 0.81 mg·L~(-1).After one hundred primers were analyzed with random amplified polymorphic DNA (RAPD),the seven different DNA fragments were produced by the N7059 strain of recombined yeasts,and,the polymerase chain reaction(PCR) verified that one of them came from the genome of G.uralensis,indicating a successful transfer of genetic information by ion implantation.     

米曲霉pyrG 基因克隆及其同源转化系统的建立

建立以乳清酸核苷-5＇- 磷酸脱羧酶基因(pyrG)为选择标志,以米曲霉为宿主菌的同源转化系统。以米曲霉基因组为模板,通过PCR 扩增获得pyrG 基因,将该片段与表达载体pMD18-T 相连,转化大肠杆菌DH 5α,经蓝白斑筛选、PCR 快速筛选、酶切和测序验证,获得重组质粒pMD-pyrG,完成pyrG 基因的克隆。序列分析表明该基因与米曲霉KBN616 的pyrG 基因编码序列的同源性为99.9%,两者经推导的氨基酸的同源性为99.6%。以米曲霉 pyrG 营养缺陷株为受体菌,通过PEG/CaCl2 诱导的原生质体转化方法,将重组质粒导入该受体菌,使米曲霉pyrG 缺陷株发生基因转化,成为pyrG+,由此成功建立了以pyrG 为筛选标记基因、同型米曲霉pyrG 基因缺陷株为受体菌的基因转化系统。     

野油菜黄单胞菌产胶基因GumD克隆与重组质粒的构建

目的:建立野油菜黄单胞菌产胶基因GumD的克隆与重组质粒构建的方法。方法:以优化PCR实验条件扩增得到野油菜黄单胞菌产胶基因GumD片断,与抗药性质粒pMD18-T连接,获得重组质粒,并用CaCl2法转化JM109大肠杆菌细胞。结果:DNA测序结果表明,成功克隆产胶基因和构建重组质粒。结论:此方法可用于克隆野油菜黄单胞菌产胶基因和构建产胶基因重组质粒。     

Human Gingival Integration-Free iPSCs; a Source for MSC-Like Cell

Mesenchymal stem cells (MSCs) are considered a potential autologous therapy for tissue engineering. The available procedures for MSC retrieval from patients are invasive, and their limited in vitro proliferation restricts their use in the treatment of damaged tissues. Therefore, it is important to establish an alternative and safe source of MSCs. The objective of this study was to demonstrate induced pluripotent stem cell (iPSC) generation from a combination of an accessible source tissue and an integration-free method; we also attempted the differentiation of iPSCs into MSC-like cells (MSLCs) for future autologous tissue engineering. iPSCs were derived from human gingival tissues, which are easily accessible in the field of dentistry, via the use of non-integrating episomal plasmids. Established iPSCs expressed embryonic stem (ES) cell-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratoma formation were formed from iPSCs, showing their capacity to differentiate into three germ layers. Furthermore, we were successful in differentiating iPSCs into MSLCs. They tested positively for their capacity of trilineage differentiation. Our results demonstrate that human gingival integration-free iPSCs, readily accessible stem cells generated using episomal plasmid vectors, are a promising source of MSLCs, which can be used in tissue regeneration.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

Integrated biological and chemical analysis of organochlorine compound pollution and of its biological effects in a riverine system downstream the discharge point

Pollution in riverine systems, along with its biological effects, may propagate downstream even at considerable distances. We analyzed the organochlorine compound (OC) pollution in a section of the low Ebro River (Northeast Spain) downstream a long-operating chlor-alkali plant. Maximal levels of OCs and of their associated dioxin-like biological activity occurred in residue samples from the plant, and persisted in river sediments some 40 km downstream (Xerta site). Biological analysis at multiple organization levels in local carp (Cyprinus carpio, EROD, Cyp1A mRNA expression in the liver, hepatosomatic index, condition factor, and micronuclei index in peripheral blood) showed a similar pattern, with a maximal impact in Ascó, few kilometers downstream the plant, and a clear reduction at Xerta. This combination of chemical, molecular, cellular and physiological data allowed the precise assessment of the negative impact of the chlor-alkali plant on the quality of river sediments and on fish, and suggests that sediments may be a reservoir for toxic substances even in dynamic environments like rivers.