邻苯二酚2,3—双加氧酶在大肠杆菌的表达与定域

本文在大肠杆菌/枮草芽孢杆菌间的穿梭质粒pTG 402的基础上构建了几个新的带有显色标志基闲xylE的表达质粒,摸索了该基因所编码的邻苯二酚2,3-双加氧酶(CatO_2ase)的表达条件,分析了该酶一级结构与二级结构的亲水性和疏水性,测定了它在大肠杆菌中的产量与分布。结果表明,CatO_2ase与各质粒的表达量不等,表达量高低与培养时间、宿主菌及诱导与否等影响因素有关;表达后有部分酶可在胞外测出,但大部分仍定域于膜内,亲、疏水性分析示该酶不具分泌性蛋白的显著特点。因该酶易于检测和定量,可作为一种选择性标记和监测指示系统在基因工程中推广应用,同时亦为用基因工程菌消除芳烃类化合物的污染提供了理论依据。     

嗜热脂肪芽孢杆菌CU21中有关质粒pFDX163的整合和游离状态的一种新的相转变现象

pFDX1是带有外源基因xyIE的重组质粒。从嗜热脂肪芽孢杆菌CU21(pFDX1)出发,提高培养温度后选择卡那霉素抗性突变菌落,得到一个变异株CU21-163。该菌株含有突变质粒pFDX163,它由一个来自宿主基因组的2.0kb的H片段插入pFDX1而构成。pFDX163可通过H片段的同源重组而整合到染色体上。CU21-163包含y、w两类细胞,二者的xyIE基因表达量有明显差异。这两类细胞在分裂过程中呈现一种新的相转变现象,即y细胞的后代中经常出现少数w细胞,w细胞的后代中经常出现少数y细胞。对CU21-163的不同细胞群体的总DNA样品中游离质粒与整合质粒的含量进行了测定,由此推断y细胞含有游离质粒和整合质粒,w细胞只含整合质粒。     

在链霉菌中表达透明颤菌血红蛋白需要异源启动子

构建了质粒pIJ4083Mpro、pIJ4083\|pro\,pWLD8和pFW3。在浅青紫链霉菌TK24中,启动子探针质粒pIJ4083上的邻苯二酚双加氧酶基因(xylE)不能被透明颤菌血红蛋白基因(vgb)的启动子带动转录,表明vgb启动子在链霉菌中无作用。TK24中,pWLD8和pFW3均能表达透明颤菌血红蛋白(VHb),pWLD8上可能是由Plac带动vgb的表达;pFW3上vgb基因去掉了非必要部分,克隆在PCR扩增得到的glnA启动子下游,两者连成嵌合基因。     

嗜热脂肪芽孢杆菌启动子克隆载体pFDC4和表达型载体pFDC11的构建

以pPL703的衍生质粒pPGV5为载体,从嗜热脂肪芽孢杆菌CU21总DNA的Sau 3A酶切产物中得到1个0.54kb的启动子片段,它能促进载体上的无启动子的cat-86基因在嗜热脂肪芽孢杆菌及枯草芽孢杆菌中表达。这一片段以正、反向插入pPGV5载体,都能使重组质粒转化CU21原生质体的效率提高10~(?)至10(?)倍。Southern杂交实验表明,这一启动子片段与Imanaka等报道的来自CU21中的隐蔽性质粒pBS02的能提高转化效率的1.6kb Eco RI片段是同源的。利用所得到的0.54kb Sau 3A片段构建了新的启动子克隆载体pFDC4和表达型载体pFDC11,二者都能以很高的效率转化CU21原生质体。     

用于丝状真菌的苯菌灵标记基因benA表达载体的构建

为了构建适用于丝状真菌遗传转化的表达载体,在质粒pAN52-1的高效组成型三磷酸甘油醛脱氢酶基因启动子PgpdA和色氨酸C基因终止子TtrpC之间加入内切酶NotⅠ和ClaⅠ两个位点,构成质粒pAN52-2;把质粒pAN52-2中从启动子PgpdA到终止子TtrpC的基因片段通过PstⅠ和XbaⅠ酶切位点导入到pUC19载体上,并且应用PCR的方法在启动子Pg-pdA的前端引入HindⅢ酶切位点,构成质粒pUCPT-2;通过PCR,在苯菌灵抗性基因benA两端分别加入内切酶NotⅠ和ClaⅠ酶切位点,并克隆到pGEM-T载体上,得到质粒pGEM-Ben;用NotⅠ分别酶切质粒pUCPT-2与pGEM-Ben,连接苯菌灵抗性基因benA到pUCPT-2上,得到了pUCPT-Ben载体。经PCR、酶切和核苷酸序列检测,证实了PgpdA-benA-TtrpC表达元件连接成功,即得到了苯菌灵抗性基因高效表达载体。     

高温α-淀粉酶高表达的研究

为了进一步提高工业上重要的地衣芽胞杆菌高温α-淀粉酶(BLA)的发酵生产性能,以高温α-淀粉酶基因(amyL)为目的基因,构建地衣芽胞杆菌高温α-淀粉酶基因产生菌的整合表达通用性质粒pB li16 s-amyL-EryR,采用原生质体转化法将此整合型表达质粒导入B.licheniformis CBBD302,在整合表达质粒中的16SrDNA序列的介导下实现了同源整合。挑选20株阳性转化子,分别通过提高培养基中红霉素的使用浓度,增加染色体上目的基因amyL的拷贝数,由此获得的重组菌的BLA生产水平提高了56.37%~80.29%。     

白地霉ch-3脂肪酶Ⅰ基因在大肠杆菌中的表达

构建了白地霉脂肪酶Ⅰ的基因工程菌,为进一步进行蛋白质工程改造和脂肪酶应用奠定了基础。从新疆昌吉市油脂化工厂含油冻土中分离得到1株低温脂肪酶产生菌-白地霉ch-3。该菌发酵上清液中的脂肪酶最适作用温度为35℃,在0℃仍可保持66%的相对酶活力。应用PCR技术从白地霉ch-3基因组DNA中克隆得到脂肪酶Ⅰ基因lip1,将该基因与原核表达质粒载体pET-22b（＋）连接,构建重组质粒pETl-ip1,转化E.coliBL21（DE3）,酶切鉴定,筛选得到重组菌。十二烷基磺酸钠-聚丙希酰胺（SDS-PAGE）显示重组脂肪酶Ⅰ的相对分子质量约为5.8×10^4,酶活为2.73 U/mL,表明lip1基因的表达产物具有正常的生物学活性。白地霉ch-3脂肪酶Ⅰ基因lip1能够在大肠杆菌中有效地表达。     

过量表达DegQ有利于地衣芽胞杆菌表达高温α-淀粉酶

研究了过量表达DegQ对地衣芽胞杆菌表达高温α-淀粉酶的影响。通过PCR从地衣芽胞杆菌基因组中扩增得到degQ基因,将其克隆到pHY—P43载体中,得到重组质粒pHY—P43-degQ。将其分别转化至地衣芽胞杆菌ATCC14580和B0204中,得到重组菌ATCC14580（pHY—P43-degQ）和B0204（pHY—P43-degQ）。通过发酵试验,证实degQ基因的表达能够显著提高高温α-淀粉酶的表达水平。     

辛德毕斯病毒E2包膜蛋白可溶性表达条件的优化

目的:通过优化表达条件,提高辛德毕斯病毒E2包膜蛋白胞外区的可溶性表达量。方法:构建含E2基因胞外区的重组表达质粒pGEX-6p-1-E2,筛选合适宿主菌和诱导温度,并构建5种分子伴侣共表达系统(即pG-KJE8、 pGro7、pKJE7、 pG-Tf2和pTf16 5种分子伴侣质粒分别与重组表达质粒pGEX-6p-1-E2共表达),筛选最适分子伴侣质粒。结果:(1)E2蛋白的表达量在E.coli BL21、BL21 (DE3) pLysS、Rosetta (DE3)及Origami B (DE3) 4种表达菌中没有明显差别;(2)16℃诱导时E2蛋白在上清中可溶性表达量最高;(3)分子伴侣质粒pG-Tf2使目的蛋白的可溶性表达量提高了15.7%,作用最为显著。结论:通过优化表达条件及使用分子伴侣共表达系统提高了E2蛋白的可溶性表达,为进一步E2蛋白的相关研究奠定了基础。     

弗氏志贺菌5a 型 M90T 株 ClpB 蛋白在大肠杆菌中的融合表达和纯化

目的：构建弗氏志贺菌cZp8基因的原核表达质粒,在大肠杆菌中表达后,纯化带T7标签的ClpB蛋白。方法与结果：PCR扩增得到线性化表达载体pET24a与2574bp的c加曰基因片段,利用不依赖连接反应的克隆法（LIC）进行克隆,得到重组质粒pET-ClpB,转入大肠杆菌BL21（DE3）中进行诱导表达,通过一系列条件优化,确定可溶性表达条件为在30℃下、用1mmol／LIPTG诱导2h;利用抗T7单克隆抗体琼脂糖珠进行亲和纯化,得到了纯度很高的相对分子质量为95×10^3的ClpB-T7融合蛋白。结论：实现了ClpB-T7在大肠杆菌中的可溶性表达,并纯化获得了高纯度的融合蛋白。     

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.