硫色曲霉木聚糖酶基因xynA与xynB在大肠杆菌中的融合表达及酶学性质分析

根据硫色曲霉木聚糖酶基因xynA和xynB序列设计引物,PCR扩增获得574bp、594bp的目的基因片段.两段DNA分别用EcoRⅠ/BamHⅠ、BglⅡ/HindⅢ酶切后,连接到载体pET-28a( )的多克隆位点,构建了xynA和xynB融合表达载体pET-xynAB,即在木聚糖酶A和B之间引入了7个氨基酸的寡肽序列(GlyGlyGlySerGlyGlyGly).将pET-xynAB转化大肠杆菌BL21,经IPTG诱导,SDS-PAGE检测到了一条约50ku的特异性表达条带.Ni-NTA柱纯化后的特异性蛋白经8mol/L脲素变性,梯度透析使蛋白质复性.酶学性质分析表明,该重组木聚糖酶的最适催化温度为50℃,最适pH值为4.4.在pH2.4～5.4范围内,酶的相对活性都在75%以上,重组酶在80℃保温30min还有近50%的残余酶活.不同种类的金属离子对重组酶的活力影响不同.     

硫色曲霉木聚糖酶基因xynA与xynB在大肠杆菌中的融合表达及酶学性质分析

根据硫色曲霉木聚糖酶基因xynA和xynB序列设计引物，PCR扩增获得574 bp、594 bp的目的基因片段．两段DNA分别用EcoRⅠ/BamHⅠ、BglⅡ/HindⅢ酶切后，连接到载体pET-28a(+)的多克隆位点，构建了xynA和xynB融合表达载体pET-xynAB，即在木聚糖酶A和B之间引入了7个氨基酸的寡肽序列(GlyGlyGlySerGlyGlyGly)．将pET-xynAB转化大肠杆菌BL21，经IPTG诱导，SDS-PAGE检测到了一条约50 ku的特异性表达条带．Ni-NTA柱纯化后的特异性蛋白经8 mol/L脲素变性，梯度透析使蛋白质复性．酶学性质分析表明，该重组木聚糖酶的最适催化温度为50℃，最适pH值为4.4．在pH 2.4～5.4范围内，酶的相对活性都在75%以上，重组酶在80℃保温30 min还有近50%的残余酶活．不同种类的金属离子对重组酶的活力影响不同．     

运用定点突变提高重组木聚糖酶在毕赤氏酵母中的表达

运用PCR介导的定点突变对米曲霉(Aspergillus　oryzae)来源的木聚糖酶在毕赤酵母中的重组表达进行了研究,获得一表达量远远高于亲本的突变株I156A,对其进行了提纯并研究其酶学特性,除热稳定性外其余与亲本基本一致。突变株I156A所产木聚糖酶XynFl的分子量为35kD,在pH 4～9范围内稳定,最适pH为70,最适温度为45℃,在50℃以下稳定性略高于亲本。     

日本曲霉产β-呋喃果糖苷酶的性质研究

目的:试验确定日本曲霉产β—呋喃果糖苷酶的酶学性质。方法:将β-呋喃果糖苷酶在不同的温度、pH值、底物浓度、反应时间的条件下测定酶活。结果:确定了日本曲霉产β-呋喃果糖苷酶的性质。日本曲霉产β-呋喃果糖苷酶分子量约为89kDa,最适反应温度为50℃,最适pH值为5.5。温度在40℃-60℃,pH在5-7范围内酶比较稳定,最适底物浓度为50%,反应时间3h-4h。结论:确定了日本曲霉产β-呋喃果糖苷酶的性质,为制取高含量的低聚果糖提供条件。     

极端耐热木聚糖酶基因在枯草芽孢杆菌中的整合表达

目的:为了在枯草芽孢杆菌中整合表达极端耐热木聚糖酶。方法:将嗜热网球菌(Dictyoglomus thermophilum)Rt46B.1的极端耐热木聚糖酶基因xynB通过穿梭载体pDL整合到B.subtilis168染色体上,使其实现表达。结果:极端耐热木聚糖基因在枯草芽孢杆菌中成功整合并表达。结论:基因工程菌B.subtilis168-xynB能外泌表达极端耐热木聚糖酶,且表达水平为0.732IU/mL,比在大肠杆菌中的高。酶学性质表明,此酶分子量约为24kD,其最适反应温度为85℃,最适反应pH值为6.5,且在弱碱性条件下稳定。     

棒曲霉22产木聚糖酶的研究

从105株霉菌和酵母菌中筛选到一株木聚糖酶活力较高的棒曲霉(Aspergillua clavatus)菌株22。该菌株适宜的产酶培养基为(g/1):蔗渣半纤维素30,NH₄NO₃ 5,酵母膏5,麸皮10,吐温801和少量无机盐,初始pH5.5。最适的孢子接种量为4.9×10⁶个/ml。在上述培养基中28℃振荡培养72h.木聚糖酶活力可达335.9u/ml。酶反应的最适温度为50℃;最适pH为4.8,在pH 6-11酶活性稳定。     

利用内源元件在谷氨酸棒杆菌中分泌表达木聚糖酶

以来自于谷氨酸棒杆菌内源AH6启动子和5′UTR及其前38 bp结合合适的Shine-Dalgarno (SD)序列,构建双顺反子表达载体对木聚糖酶进行表达。为了能够实现分泌表达,选取了来自谷氨酸棒杆菌的两种分泌途径的信号肽,分别为Tat型的CgR0949及Sec型的CspB信号肽。在实现分泌表达之后,对其进行5 L发酵罐的扩大培养以提高分泌量。并对纯化的木聚糖酶进行了部分酶学性质的研究,包括最适催化pH及酸碱耐受性;最适催化温度及热稳定性。结果表明:在上述表达体系中,以CgR0949为信号肽木聚糖酶不能分泌到胞外;木聚糖酶能在CspB信号肽的引导下分泌到胞外,分泌表达量为486.2 U/mL。木聚糖酶的分泌量在5 L发酵罐水平上达到1 648.7 U/mL,是摇瓶培养的3.4倍。该木聚糖酶的最适反应pH为4.5,最适温度为45℃;在pH 4–11范围内4℃处理24 h酶活保持在80%以上;在50℃前处理15 min酶活保持在95%以上,超过60℃则酶活迅速下降至20%及其以下。上述结果表明,谷氨酸棒杆菌内源元件能有效用于木聚糖酶的分泌表达,扩大培养能进一步提升木聚糖酶的分泌量。该双顺反子表达体系能为外源蛋白在谷氨酸棒杆菌中的分泌表达提供一种可用的工具。此外,通过酶学性质的研究可进一步提高木聚糖酶的催化效率。     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株,其中编号为QH14的菌株产酶量达648.79U/mL,纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌,命名为Bacillus sp.QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14,并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃,最适pH为9.2;55℃处理1h仍保持50%的活力;在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力,且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活,显示了该碱性木聚糖酶较好的热稳定性和碱稳定,提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

嗜酸青霉酸性木聚糖酶产生特性及部分酶学性质

从煤矿酸性废水中分离到一株产木聚糖酶青霉,通过酸性液体培养研究了菌体生长对pH的响应及木聚糖酶的产生特征,并测定了木聚糖酶的部分应用性质.结果表明:实验菌株嗜酸,菌丝生长最适pH为2.0,孢子萌发生长适宜pH为3.0～4.0;木聚糖诱导菌体在生长稳定期大量产生木聚糖酶,蛋白胨是菌体产酶的适宜氮源;菌株所产木聚糖酶属于酸性木聚糖酶,反应最适pH 3.5、最适温度50 ℃～55 ℃,pH 2.0时酶活达到最高活力的72%,在最适反应条件下保温60 min,残余酶活接近70%,适用于较强酸性的高温加工环境.     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株, 其中编号为QH14的菌株产酶量达648.79 U/mL, 纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌, 命名为Bacillus sp. QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14, 并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃, 最适pH为9.2; 55℃处理1h仍保持50%的活力; 在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力, 且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活, 显示了该碱性木聚糖酶较好的热稳定性和碱稳定, 提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

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.