2,5-DKG还原酶Ⅰ的分离纯化与性质研究

欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸（2,5-DKG）还原酶Ⅰ基因,5L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE—Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶Ⅰ,纯化了5倍,得率27％,比活力为3,418U／mg。测定了该酶的一些特性参数：分子量为34kD,等电点为6．0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙酸（2-KLG）,对NADPH和2,5-DKG底物的Km值分别是0．29mmol／L和14．7mmol／L,1mmol／L Cu^2＋、Zn^2＋等有强烈抑制作用,EDTA和巯基乙醇对该酶没有抑制作用,酶的最适pH为7．0,最适反应温度为40℃。     

2,5-DKG还原酶I的分离纯化与性质研究*

欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸(2,5-DKG)还原酶I基因,5 L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE-Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶I,纯化了5倍,得率27%,比活力为3,418 U/mg。测定了该酶的一些特性参数:分子量为34 kD,等电点为6.0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙     

棒状杆菌2，5-DKG还原酶基因在欧文氏菌中的表达

将能够在大肠杆菌内高效表达棒状杆菌2,5-DKG还原酶I基因的质粒pBL4改造成为具有链霉素抗性的质粒pBLS,采用改进的感受态转化法将pBLS导入能够利用葡萄糖高产2,5-DKG的欧文氏菌SCB125中,通过提高温度诱导,经SDS-PAGE分析2,5-DKG还原酶I获得了高效表达,占菌体总蛋白的22%,不形成包涵体。体外酶活测定结果表明表达的酶具有较高的活力。同时,通过凝胶活力染色发现了宿主欧文氏菌SCB125中存在一个活力较强的2-酮基醛糖还原酶。     

酵母3-脱氧葡糖醛酮代谢酶的分离纯化及部分性质

3-脱氧葡糖醛酮 ( 3- deoxyglucosone)是美拉德反应的主要中间产物 ,对生物体具有毒性作用 .用硫酸铵分部沉淀、DEAE- cellulose52、Hydroxyapatite、DEAE- Sepharose CL- 6B柱层析从酿酒酵母 YBr-M( S.cerevisiae YBr-M)抽提液中分离纯化了 3-脱氧葡糖醛酮代谢酶 (以 NADPH为辅酶 ) .该酶是单一的分子 ,分子量为 44k D,反应最适 p H为 7.0 ,p H6.0～ 8.0之间酶活性相对稳定 ,以 3-脱氧葡糖醛酮为底物的米氏常数 Km 为 2 .2 5mmol/ L.在 35℃以下保温 30 min酶活不变 ,50℃保温 30 min后酶活损失 50 % .该酶对二羰基化合物的活性较高 ,对单羰基化合物则较低 ,其催化作用受碘乙酸、N-乙基顺丁烯二酰亚胺的抑制 ,而被β-巯基乙醇、二硫苏糖醇激活 ,催化作用必须以 NADPH为专一辅酶 ,当用 NADH代替 NADPH时 ,活力只有 5.3% .     

2,5-DKG还原酶Ⅱ基因的克隆和在大肠杆菌中的表达*

参照文献上的2,5-二酮基-D-葡萄糖酸（简称2,5-DKG）还原酶II基因序列,合成两个引物序列并在两端加上EcoRI和BamHI两个酶切位点,抽提棒状杆菌SCB3058菌株的染色体为模板进行PCR反应,克隆得到2,5-DKG还原酶II基因,酶切验证与预期的结果相符合。将此片段克隆到pGEM-T载体上保存．将2,5-DKG还原酶II基因用EcoRI和BamHI内切酶切下,连接到pBV220载体上,构建成表达载体。42℃诱导不能得到稳定的蛋白表达条带和酶活力,测序发现基因的3’末端的原PCR引物外少合了一     

棒状杆菌2,5-二酮基-D-葡萄糖酸还原酶Ⅰ基因在大肠杆菌中的克隆与表达

从棒状杆菌(Corynebacterium sp.SCB3058)初步纯化得到两个2,5-二酮基-D-葡萄糖酸(2,5 DKG)还原酶,在此基础上利用PCR技术,以基因组DNA为模板,扩增得到含有2,5-DKG还原酶Ⅰ基因的片段,定向连接到PGEM3Zf(+)并转化大肠杆菌DH5α,筛选得到阳性克隆pGEM813.序列分析表明,片段全长1107bp,含有一个834bp的开放阅读框架,编码产生由278个氨基酸组成的分子量为34kD的蛋白.将目的基因的调控序列进行缺失突变后,克隆到原核表达载体pBL上获得表达质粒pBL4.通过温度诱导,经SDS-PAGE分析有明显的表达条带,约占菌体总蛋白的20%,并且表达的蛋白具有较高的酶活力.构建的优良基因工程菌为最终实现从葡萄糖一步发酵产生维生素C前体2-酮基-L-古龙酸打下了基础.     

2,5-DKG还原酶Ⅱ基因的克隆和在大肠杆菌中的表达

参照文献上的2,5-二酮基-D-葡萄糖酸（简称2,5-DKG）还原酶II基因序列,合成两个引物序列并在两端加上EcoRI和BamHI两个酶切位点,抽提棒状杆菌SCB3058菌株的染色体为模板进行PCR反应,克隆得到2,5-DKG还原酶II基因,酶切验证与预期的结果相符合。将此片段克隆到pGEM-T载体上保存．将2,5-DKG还原酶II基因用EcoRI和BamHI内切酶切下,连接到pBV220载体上,构建成表达载体。42℃诱导不能得到稳定的蛋白表达条带和酶活力,测序发现基因的3’末端的原PCR引物外少合了一个碱基,终止密码子发生移码突变而消失。此外在5’端的启始密码子ATG前有三个碱基与pBV220载体上的SD序列发生配对。据此,重新设计和合成了PCR引物,并用pBV220和pBL4载体构建了两个表达载体。42℃诱导表达均得到了稳定的表达条带和较高的酶活力．     

细菌3-脱氧葡糖醛酮代谢酶的纯化及性质研究

细菌Bacillus sp.2粗酶液通过(NH₄)₂SO₄分级分离、Q Sepharose FF、Sephadex G-100(Ⅰ)、Hydroxyapatite和Sephadex G-100(Ⅱ)柱层析分离,纯化了一种以NADPH为辅酶的3-脱氧葡糖醛酮(3-DG)代谢酶,定性为2-羰基醛还原酶.纯化酶的比活力为63.75 U/mg,在SDS-聚丙烯酰胺凝胶上显示一条蛋白质带.该酶分子质量约为32 ku,酶反应最适pH约为6.2, 在pH 5～8, 温度25～30℃之间酶保持稳定;该酶对3-DG的K_m为2.3 mmol/L.添加适量的EDTA、巯基乙醇或二硫苏糖醇能明显提高酶的活性;而碘乙酸、N-乙基顺丁烯二酰亚胺抑制酶的活性.     

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

枯草芽孢杆菌WHNB02植酸酶的酶学性质研究

从118份样品中分离到1株产植酸酶的枯草芽孢杆菌(Bacillus subtilis,WHNB02),其发酵液经乙醇沉淀、硫酸铵分级沉淀及Sephadex G-100柱层析等步骤后分离纯化了该酶,纯化倍数约为31.5倍,回收率为13.0%。该酶为单体酶,SDS-PAGE测得的分子量约为43ku,以植酸钠为底物的Km值为0.5mmol/L,酶反应的最适温度为60℃,80℃作用10min酶活保存61%,最适pH为7.0,在pH6.0~10.0范围内稳定,酶活性及稳定性都需Ca2 存在。EDTA、Mn2 、Ba2 (5mmol/L)对酶活具有很大的抑制作用。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.