耐热糖化酶基因的克隆及其在枯草杆菌中的表达*

应用PCR技术从嗜热古生菌硫磺矿硫化叶菌(Sulfolobussolfataricus)中扩增到大小约为1.9kb编码嗜热糖化酶的DNA片段,并将其插入枯草杆菌诱导型表达载体pSBPYF,获得含有该糖化酶基因的重组质粒pSGAYF,转化枯草芽孢杆菌DB1342。经蔗糖诱导后,该糖化酶在枯草杆菌DB1342(pSGAYF)中获得胞外分泌表达,酶活为3.6U/mL,最适温度为90℃,最适pH6.0。     

嗜热糖化酶基因在重组黑曲霉工程菌中的表达及酶学性质初步研究

为了提高糖化酶的耐热性能,降低淀粉糖化发酵工艺的生产成本,构建了同源整合载体pEasy-glaAdir以及pEasyssg,将黑曲霉(Aspergillus niger)的糖化酶基因(glaA)灭活,并将硫磺矿硫化叶菌(Sulfolobus solfataricus)的嗜热糖化酶基因(ssg)插入到黑曲霉基因组中,筛选得到表达嗜热糖化酶的重组黑曲霉工程菌(A.nigerWW1)。重组菌的发酵结果显示,嗜热糖化酶在黑曲霉中得到了分泌表达,发酵液酶活达到3 030 U/mL。重组嗜热糖化酶的最适反应温度为90℃,最适pH为6.0,该酶具有较高的热稳定性,在80℃时的半衰期在60 min以上,具有良好的工业应用前景。     

嗜热脂肪芽孢杆菌中耐热蛋白酶基因的克隆

用鸟枪法把嗜热脂肪芽孢杆菌313一l(供体菌)染色体上的蛋白酶基因克隆到质粒pPL603上,并在枯草杆菌中得到表达。此基因位于6．6kb的EcoRI酶切片段上,带有重组质粒的枯草杆菌所产生的蛋白酶活性比供体菌株约提高30倍左右。该酶的最适反应温度为75℃,最适pH为7．0左右,是一个中性蛋白酶,在80℃作用30min后仍保留85％以上的酶活。     

嗜铬粒蛋白N端片段基因在枯草杆菌中的表达及表达产物的活性分析

嗜铬粒蛋白(CGA)是存在于分泌细胞的由439个氨基酸组成的可溶性蛋白。近年的研究发现CGA的N端具有抗血管收缩、抗细菌和抗真菌的功能。为了寻找高效低毒的抗真菌片段,利用PCR技术扩增了编码人嗜铬粒蛋白N端1-76位氨基酸（CGA1-76）的DNA片段,并将之克隆进本实验构建的枯草杆菌诱导型表达载体pSBPTQ,获得含CGA1-76基因的重组质粒pSVTQ,转化蛋白酶三缺陷的枯草杆菌DB403。经蔗糖诱导后,CGA1-76片段在枯草杆菌DB403（pSVTQ）中获得表达,产物分泌到细胞外,分泌量约为5mg/L,占总分泌蛋白的133% 。测试了表达产物对几种丝状真菌和酵母的抑制作用,发现在4μmol/L的浓度下,枯草杆菌表达的重组CGA1-76对镰刀菌、链格孢霉及白假丝酵母有明显的抑制作用。     

嗜热脂肪地芽孢杆菌NAD激酶克隆表达及酶学性质研究

NAD激酶能催化NAD生成NADP。本研究采用PCR技术从嗜热脂肪地芽孢杆菌基因组中获得NAD激酶基因,以pET30a（＋）为表达载体、E．coliBL21（DE3）为宿主菌,实现其在大肠杆菌中异源表达,并进行酶学性质研究。结果显示,嗜热脂肪地芽孢杆菌中NAD激酶编码基因大小为816bp,酶分子量大约为35kD。酶学性质分析表明,来源于嗜热脂肪地芽孢杆菌的NAD激酶最适反应温度和pH分别为35℃、pH7．5,在35qC中保温2h后仍能保持80％左右的活性。Mn2＋、Ca2＋对该酶有较强的激活作用,在最适反应条件下该酶的比活力为4．43U／mg。动力学性质分析结果显示NAD激酶对底物NAD催化的k和圪。,分别为1．46mmol／L和0．25tzmol／（L·min）。NAD激酶在大肠杆菌的异源表达为以NAD为底物生物合成NADP提供了更多生物资源。     

嗜盐微生物

高盐环境通常是指那些盐浓度高于海水的环境．在这些环境中能够生存的微生物可划分为三类：一“类是能耐受一定浓度的盐溶液,但在无盐存在条件下生长最好的菌称为耐盐菌．第二类是一定浓度的盐为菌体生长所必需,且在一定浓度的盐溶液中生长最好,称为嗜盐菌．在盐浓度从零至饱和的盐溶液中均能生长,在一定浓度的盐溶液中生长最好的特殊类群称为多能盐生苗。依据嗜盐浓度的不同,嗜盐菌又可分为轻度嗜盐菌（最适盐浓度0．2—0．smol／L）、中度嗜盐菌（最适盐浓度0．5—2．omol／L）和极端嗜盐菌（最适盐浓度＞3mol／U,其中部分极端嗜…     

利用degQ基因提高枯草芽孢杆菌纤溶酶表达

从枯草芽孢杆菌(Bacillus subtilis)中通过PCR扩增得到degQ基因,将其克隆到含有枯草杆菌纤溶酶基因的蔗糖诱导表达载体pUBS中,并转化至B．subtillis DB403受体菌,得到基因工程菌DB403(pUBSD)。通过发酵表达证实degQ基因能增强枯草杆菌纤溶酶的表达,酶活提高了2．2倍。同时还对不同种类的糖、不同浓度蔗糖、不同诱导时间等发酵条件进行优化和比较研究。     

中性蛋白酶基因诱导型表达分泌载体的构建

利用PCR方法分别扩增出sacB基因的启动子-信号肽序列（sacR）和枯草芽孢杆菌中性蛋白酶的前肽-成熟肽序列,将两者连接后克隆入载体pHP13中,构建了含有中性蛋白酶基因的诱导型表达分泌载体pHP13SN,再将其转化入枯草杆菌DB104,获得基因工程菌DB104(pHP13SN)。中性蛋白酶基因在蔗糖的诱导和sacR的调控下实现了分泌表达,并获得了具有生物学活性的中性蛋白酶。     

嗜热毛壳菌纤维素酶（CBHⅡ）cDNA的克隆及在毕赤酵母中的表达

嗜热毛壳菌Chaetomium thermophilum CT2是一种土壤腐生菌,可产生具有重要工业生产价值的纤维素酶类。RACE-PCR获得嗜热毛壳菌纤维二糖水解酶Ⅱ（CBHⅡ）的编码基因(cbh2)。DNA序列分析表明cbh2的开放阅读框由1428个碱基组成,编码476个氨基酸。推断的氨基酸序列包含一个典型真菌纤维素酶的糖结合域（CBD）、催化域（CD）以及二者之间富含脯氨酸和羟基氨基酸的连接桥。根据氨基酸序列推算该酶分子量为53kD,属于糖苷水解酶第六家族,具有该家族催化保守区的典型特征。PCR扩增cbh2的成熟蛋白编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶蛋白的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.2 mg/mL。经硫酸铵沉淀、DEAESepharose Fast flow阴离子层析等步骤纯化了该重组表达蛋白。SDS-PAGE得到重组蛋白分子量为67kD,与从嗜热毛壳菌中纯化的该酶分子量一致。该重组纤维二糖水解酶作用的最适合温度50℃,最适pH4.0,在70℃的半衰期为30min,具有较好的热稳定性。     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

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.