重组人白细胞介素6部分生化物理学性质的鉴定

采用工程菌 E.coli DH5 α(p IB- h IL- 6 ) ,经过发酵培养、表达和包含体的分离纯化等步骤 ,获得了 N端缺失 5个氨基酸的 rh IL- 6纯品。对其理化生物学性质进行分析。结果表明 ,纯品 N-端氨基酸序列为 MEDSKD… ;在 2 76 nm波长处有一个特异的蛋白吸收峰 ;分子量为 2 1 487.2 ;裂解的肽段分布在理论值的肽段分布范围之内 ;p I为 6 .1 ;能与兔抗人 IL- 6抗体特异性结合 ;比活性达2 .0 5× 1 0 8IU/mg。     

重组人白细胞介素6的制备

我们采用本院基础医学研究所组建的ＩＬ-６工程菌Ｅ．ｃｏｉｌＤＨ５ａ（ｐＢＶ-ｈｌＬ－６）,在选定的培养基及ｐＨ值下,采用３０升发酵罐进一步观察了工程菌的生长和ｒＩＬ－６的表达,确定了发酵工艺条件。在此条件下连续进行了三批次的培养试验。结果表明,工程首的生长密度达到２．５１±０．０２ｇ［干重］／Ｌ［发酵液］,ｒＩＬ-６产率为１８２．４±２．０ｍｇ／ｇ［干重］。ｒＩＬ-６以包涵体形式表达于大肠杆菌细胞中,破菌后选用非离子型去垢剂或尿素等变性剂提取包涵体中的杂蛋白,可使ｒＩＬ-６的纯度达到７０．１±１．３％,收率为７１．９±１．９％。洗涤后的包涵体,经过凝胶柱纯化和复性,ｒＩＬ-６纯度达到９５％以上,柱纯化的收率为７２．３±０．９％;采用依赖ＩＬ-６,小鼠杂交瘤细胞系７ＴＤ１及ＭＴＴ比色法测定生物活性,ｒＩＬ-６比活性达２×１０８Ｕ／ｍｇ。     

重组人白细胞介素-6的纯化

重组人白细胞介素－６（ｒｈＩＬ－６）在工程菌ｐＢＶ２２０／ｒｈＩＬ－６／ＤＨ５ａ中以包涵体形式高效表达。ｒｈＩＬ－６经过工程菌体破碎、包涵体分离及抽提、复性、色谱分离后得到高度纯化。纯化产物纯度９５％,具有良好的生物学活性     

大肠杆菌表达重组人白细胞介素-9的纯化及部分性质

表达重组人白细胞介素－９（ｒｅｃｏｍｂｉｎａｎｔｈｕｍａｎｉｎｔｅｒｌｅｕｋｉｎ－９ｒｈＩＬ－９）的大肠杆菌经破碎、包涵体洗涤、裂解提取、凝胶过滤和离子交换色谱分离,得到了电泳纯的ｒｈＩＬ－９,回收率６６％,分子量与理论值相符,有刺激小鼠骨髓巨核系集落生成的活性．为ｒｈＩＬ－９的大规模制备及更深入的研究奠定了基础     

白细胞介素6重组逆转录病毒载体的构建及鉴定

目的 构建特异性过表达大鼠IL-6基因的重组逆转录病毒载体,并在大鼠嗜铬细胞瘤PC12细胞和人胚肾HEK293细胞中检测IL-6的表达。方法以大鼠骨髓间充质干细胞mRNA为模板,经PCR获得目的基因IL-6,将其定向克隆到逆转录病毒载体pSEB-3H中,构建重组逆转录病毒质粒pSEB—IL-6,经脂质体分别转染到PC12细胞和HEK293细胞中,应用Real—timePCR和ELISA的方法在mRNA和蛋白质水平检测IL-6的表达变化。继而用HEK293细胞中包装获得的含有pSEB—IL-6的病毒颗粒进一步感染PC12细胞,Real—timePCR检测,IL-6mRNA的表达水平变化。结果PCR电泳及酶切鉴定证实目的基因正确克隆至逆转录病毒载体中,其基因序列与Genbank报道一致;Real—timePCR和ELISA结果均显示,逆转录病毒质粒pSEB—IL-6转染PC12细胞和HEK293细胞后,IL-6的表达水平较对照组显著上调;经pSEB—IL-6逆转录病毒颗粒感染的PC12细胞中,IL-6mRNA表达水平较对照组提高4倍。结论成功构建了特异性表达大鼠儿-6基因的重组逆转录病毒载体pSEB—IL-6,并获得了具有感染能力的逆转录病毒颗粒,感染真核细胞后可高表达IL-6,为进一步研究IL-6的功能及其在多种疾病中的免疫调节机制提供重要的分子手段。     

人白细胞介素6cDNA克隆的分离与鉴定

我们从重组的人α干扰素处理的单层HeLa细胞常规提取Poly(A)~+RNA作为逆转录合成cDNA第一链之模板,用引物-适配接头法在噬菌质粒pTz19R中构建cDNA文库。以~(32)p-标记的480bpIL-6cDNA片段作探针进行菌落原位及狭缝印迹杂交,筛选出6个阳性克隆。其中两个克隆并用限制酶切分析及DNA序列测定做进一步鉴定。结果证明,一个克隆的cDNA片段长1.3kb,含有人白介素6全长编码区;另一个的cDNA插入片段为0.9kb,缺乏信号肽及成熟IL-6N端30个残基的编码序列。     

毕赤酵母表达重组人白细胞介素11的纯化与鉴定

报道了毕赤酵母表达人白细胞介素11的下游工艺研究,并对其产物进行了分析鉴定。所用工艺流程为离心收集上清、超滤浓缩脱盐、离子交换层析、疏水层析、凝胶过滤。所得产物经SDSPAGE电泳、RPHPLC分析、N端和C端序列分析、质谱、等电点分析和生物学活性分析,结果表明：产品纯度大于97%,结构和性质与E.coli融合表达的Neumega完全一致。     

重组人白细胞介素2复性条件的研究

由E.coli表达的重组白细胞介素2(rIL—2)以包涵体形式存在于工程菌胞浆中。用变性剂将包涵体溶解后得到的还原态rIL—2没有生物活性,必须对其进行复性才能最终得到有用的产品。本实验研究了以盐酸胍(GUHCl)为变性剂条件下,变性剂浓度、复性剂、pH值以及温度和时间等对rIL—2复性的影响。     

Relationship between physical,biochemical and biomechanical properties of tissue-engineered cartilage-carrier-constructs

Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. Physical parameters (height and weight) correlated very well with total DNA content (r²=0.86, re. 0.94). The relation between DNA content and glycosaminoglycan content was less but still significant. No significant relationship was found between the elasticity module and the DNA content, even if the elasticity module increased slightly at higher DNA content. With respect to later implantation, selection of a construct for implantation based on the weight, which can be determined non-invasive and under sterile conditions, seems to be justifiable.Revisions requested14 October 2004; Revisions received 7 December 2004     

Polymerized structure and improved physical and biochemical material properties of carboxymethyl dextran-conjugated collagen

Pepsin-solubilized collagen (PSC) was conjugated with carboxymethyl dextran (CMD) using cyanogen bromide to obtain a PSC-CMD film having improved physical properties, physiological properties, and cell affinity. The conjugation was confirmed by the loss of the alpha- and beta-subunit chains and the polymerized band on SDS-PAGE, and by a decrease in the isoelectric point to 3.2. PSC-CMD had a large polymerized structure with the 6 PSC and 228 CMD molecules. PSC-CMD was readily soluble in water, reconstructed a matrix with a less-ordered structure and a characteristic morphological shape, and lost platelet aggregation-inducing ability. The PSC-CMD film, cross-linked by ultraviolet irradiation, exhibited reduced solubility, moderate water vapor permeability, and increased flexibility. PSC-CMD coatings exhibited good cell attachment and growth for fibroblasts and vein endothrical cells.     

Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage

The enzymatic processes underlying the degradation of aggrecan in cartilage and the corresponding changes in the biomechanical properties of the tissue are an important part of the pathophysiology of osteoarthritis. Recent studies have demonstrated that the hexosamines glucosamine (GlcN) and mannosamine (ManN) can inhibit aggrecanase-mediated cleavage of aggrecan in IL-1-treated cartilage cultures. The term aggrecanase describes two or more members of the ADAMTS family of metalloproteinases whose glutamyl endopeptidase activity is known to be responsible for much of the aggrecan degradation seen in human arthritides. In this study we examined the effect of ManN and GlcN on aggrecanase-mediated degradation of aggrecan induced by IL-1alpha and the corresponding tissue mechanical properties in newborn bovine articular cartilage. After 6 days of culture in 10 ng/ml IL-1 plus ManN, mechanical testing of explants in confined compression demonstrated that ManN inhibited the IL-1alpha-induced degradation in tissue equilibrium modulus, dynamic stiffness, streaming potential, and hydraulic permeability, in a dose-dependent fashion, with peak inhibition ( approximately 75-100% inhibition) reached by a concentration of 1.35 mM. Aggrecan from explants cultured in IL-1 was found by Western analysis to be almost entirely processed down to the G1-NITEGE(373) end product. Addition of ManN or GlcN was found to produce 75-90% inhibition of this cleavage, but the proportion of aggrecan remaining in the tissue which was cleaved at aggrecanase sites in the chondroitin sulfate (CS)-rich region (Glu(1501) and Glu(1687)) was higher than with IL-1 alone. This result suggests that the preservation of mechanical properties by hexosamines in explants is primarily due to inhibition of cleavage at the Glu(373) site in the interglobular domain. While the precise mechanism by which hexosamines function in this system is unclear, the present analysis suggests that the mechanical properties examined may be predominantly a function of electrostatic repulsion due to the charged CS chains in the tightly packed repetitive sequences of the CS-1 region.     

rhlL-6影响巨噬细胞内钙离子浓度和空间分布

使用重组人白细胞介素刺小鼠腹腔巨噬细胞,应用荧光分光光度计和激光扫描共焦显微镜检测胞浆内游离钙离子的时空分布。静息状态巨噬细胞Ｃａ＾２＋浓度１０＾－８,－１０＾７ｍｏｏ／Ｌ,加入１００ｕ／ｍｌｒｈＩＬ－６后,未见作用。     

rhlL-6影响巨噬细胞内钙离子浓度和空间分布

使用重组人白细胞介素６（ｒｈＩＬ－６）刺激小鼠腹腔巨噬细胞,应用荧光分光光度计和激光扫描共焦显微镜检测胞浆内游离钙离子的时空分布。静息状态巨噬细胞Ｃａ２＋浓度１０－８｀～１０－７ｍｏｏ／Ｌ,加入１００ｕ／ｍｌｒｈＩＬ－６后,未见作用。从２００ｕ／ｍｌ至４００ｕ／ｍｌ不同浓度的ｒｈＩＬ－６则使细胞内游离Ｃａ２＋浓度呈尖峰状迅速升高３００％左右,然后急剧下降至静息值。且各个浓度对Ｃａ２＋的升高幅度及峰形基本一致,但随浓度的升高,Ｃａ２＋浓度达到高峰所需的时间逐渐缩短。这说明,胞外ＩＬ－６浓度存在着一阈值,只有达到或超过这一阈值,才能通过一定机制造成细胞内Ｃａ２＋的升高     

Determination of the biochemical properties of full-length human PIF1 ATPase

The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg²⁺ and Mn²⁺ but not Ca²⁺ and Zn²⁺. Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.     

Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase

The level of O6-methylguanine-DNA methyltransferase activity in a human cell line carrying a 1.1-kilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Mer+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from O6-methylguanine and O4-methylthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.     

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.