肉碱脱水酶突变株的获得及其休止细胞反应条件的优化

对一株产肉碱脱水酶的菌株用溴化乙锭进行诱变,并经肉碱选择性培养基进行筛选,获得了一株高活性酶活的菌株,并进行了休止细胞转化巴豆甜菜碱为Ｌ－内碱的反应条件的研究。实验确定休止细胞的最适反应温度为３０℃～３２℃,ｐＨ８．１、０．０１ｍｏｌ／Ｌ磷酸缓冲液,时间８．５～１０ｈ。最适底物浓度为６０ｍｇ／ｍｌ,最适休止细胞浓度为６０ｍｇ／ｍｌ（湿重）,产率４０％（摩尔比）。建立了休止细胞酶反应的表观米氏方程,     

微生物酶法转化生产L-肉碱的研究进展

L -肉碱作为一种新型的营养强化剂和临床药物 ,广泛应用于医疗、保健、食品等领域。L- 肉碱的生产方法有化学合成、微生物发酵、微生物酶法转化等 ,其中微生物酶法转化被认为是一种最经济且最有前途的方法。就 3种酶法转化 (DL -肉碱衍生物的酶法拆分、巴豆甜菜碱的酶法转化、D- 肉碱的酶法转化 )的微生物产酶菌株、产酶条件和酶法转化的最适条件作一概述。     

一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh 的克隆、表达及酶学性质

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。     

紫外分光光度法测定肉碱转化液中巴豆甜菜碱的含量

通过对巴豆甜菜碱和L－肉碱在紫外188－215mm的光吸收的比较,建立了紫外分光光度法测定肉碱转化液中巴豆甜菜碱含量的方法,测定波长205mm,线性范围0－25ug/ml,该方法快速方便 ,重复性好,可用于L－肉碱生产过程中巴豆甜菜碱的跟踪检测。     

Citrobacter freundii休止细胞催化合成L-多巴

以在L-酪氨酸诱导下高效表达酪氨酸酚解酶的菌株Citrobacter freundii 48003-3的休止细胞为生物催化剂,以邻苯二酚、丙酮酸钠、醋酸铵为前体,选择性合成L-DOPA。研究了反应温度、pH和前体浓度等对合成L-DOPA的影响。最优反应条件下,反应12h,L-DOPA的量可达到9．5g／L。     

植物资源开发利用新信息——国外有关植物的专利摘要(十一)

301.诱导巴豆发生不定芽的培养基 据日本1990年专利记载,诱导巴豆(Croton su-blyratus或C.joufra)产生不定芽的培养基,应含有植物激素(0.2—10ppm细胞分裂素及0.001—4ppm8-羟基喹啉(oxine)或0.1—10ppm细胞分裂素及0.005—0.5ppm赤霉素)。 302.繁殖大豆不定芽的培养基 据日本1990年专利记载,含有0.25—1.5倍无机盐、维生素(如Gamborg B_50.1—10mg/L,盐酸硫胺素及0.5—10mg/L烟酸),植物激素(如0.5—3mg/L 2,4-D)和糖的MS培养基,特别适用于大豆未成熟芽。     

利用赭曲霉NG1203羟基化齐墩果酸的研究

考察了利用赭曲霉Aspergillus ochraceusNG1203进行C11α-羟基化齐墩果酸生化反应的条件。得出了菌株摇瓶培养最佳培养基配方(g.L-1):葡萄糖20,玉米浆25,酵母膏3,K2HPO41.5,pH 6.0。菌株培养20 h,加入2 mg.L-1的齐墩果酸利于诱导羟基化酶的产生。菌株在28℃下以150 r.min-1振荡培养24 h,加入底物的乙醇溶液,使转化液中齐墩果酸的初始质量浓度达100 mg.L-1,转化液中乙醇体积分数最终达3%。经96 h转化,齐墩果酸转化率可达到10.12%。通过HPLC1、H NMR和13C NMR分析,结果表明产物为C11α-羟基齐墩果酸。     

沙枣细胞悬浮培养体系的建立

分别以沙枣幼嫩叶片,茎段为外植体诱导沙枣愈伤组织,选择生长旺盛的松散愈伤组织进行细胞悬浮培养,探讨了培养基种类和激素组合对其产生的影响.结果表明,最适的沙枣愈伤组织诱导培养基为NT(包括0.1 mg·L-1BA和0.1 mg·L-1TDZ);沙枣悬浮细胞在MS,NT,WPM,B5和IS培养基中均可生长,其中在NT培养基中生长状态最好,呈现生长均一的绿色疏松状态.激素组合对沙枣细胞生长影响很大,其中附加BA 0.1 mg·L-1和TDZ 0.1 mg·L-1组合的NT液体培养基可获得大量繁殖速度快、生长均匀一致的悬浮细胞,细胞增长系数达5.73.HPLC测定结果证明沙枣细胞中含有一定量的浓缩鞣质达5.2022 mg/gDW,这为利用沙枣悬浮细胞生产次生代谢产物提供了一条有效途径.     

大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。     

荔枝胚性愈伤组织诱导和离体保存条件的筛选

对影响荔枝(Litchi chinensis Sonn.)胚性愈伤组织诱导的因素(防褐化剂种类、花期、荔枝品种以及外植体取材部位)进行了比较研究,并对其离体保存条件进行了筛选.结果表明,在愈伤组织诱导培养基(含2.0 mg·L-12,4-D、0.5 mg-L-1NAA、30 g·L-1蔗糖和6 g·L-1琼脂的MS培养基,pH 5.8)中添加防褐化剂水解乳蛋白(0.4g·L-1),可使花药胚性愈伤组织的诱导率达到20.51%;以茎段、叶柄和幼叶为外植体,不能诱导出胚性愈伤组织,而采用花药和幼果培养,可诱导出胚性愈伤组织.其中,第1期雄花花药是最适宜的培养材料,胚性愈伤组织的诱导率可达20.11%;荔枝不同品种间幼果的胚性愈伤组织诱导率存在差异,品种‘及第'的诱导率最低.在15℃条件下,将荔枝胚性愈伤组织保存在添加20 g·L-1甘露醇的保存培养基(含1.0 mg·L-1 2,4-D、30 g·L-1蔗糖和6 g·L-1琼脂的MS培养基,pH 5.8)中,保存效果最佳,可将继代时间延至100 d.     

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.