肠球菌肽脱甲酰基酶在大肠杆菌BL21(DE3)中高效表达及其活性检测

肽脱甲酰基酶 (peptidedeformylase ,PDF)存在于所有原核生物中是其生长、代谢、繁殖必不可少的关键酶 ,但不存在于人类与其他哺乳动物细胞内 ,因而被视为新一代广谱抗生素药物筛选的理想靶点。将肠球菌 (Enterococcusfaecium)肽脱甲酰基酶基因连接到高效蛋白表达载体pET 30 A( )中 ,并转入宿主大肠杆菌BL2 1 (DE3)中进行诱导表达。在该基因的诱导表达中 ,采用不同表达条件进行诱导表达 ,最终获得表达效率极高且可溶的肽脱甲酰基酶。从宿主细胞中提取分离该酶 ,并进行酶活性检测 ,诱导表达的肽脱甲酰基酶有很高的酶活性     

死亡肽基因的合成及在大肠杆菌中的表达

将人工合成的寡核苷酸片段,通过PCR扩增得到死亡肽（thanatin）基因,并将其克隆到表达载体pGEX-3X中,序列分析结果正确。经IPTG诱导,在大肠杆菌BL21中进行高效可溶性表达,表达量可达20%以上。融合蛋白通过GST亲和层析纯化,用肠激酶酶解表达产物,用Sephadex G-25初步纯化得到具有抗菌活性的死亡肽。     

hTNFR1在大肠杆菌中可溶性表达及纯化

将人源肿瘤坏死因子Ⅰ型受体（hTNFR1）基因克隆到pET-22b表达载体,成功构建了重组表达质粒pETH1,电转到Escherichia coli BL21（DE3）表达菌株中进行摇瓶发酵。实现了hTNFR1在大肠杆菌表达系统中的重组表达。但目的蛋白全部以包涵体的形式存在于沉淀中。为了提高hTNFR1在大肠杆菌中的可溶性表达,融合标签和分子伴侣两种策略被实施用于辅助hTNFR1的可溶性表达。结果表明,在hTNFR1的N端融合NusA标签后,hTNFR1的可溶性有一定提高;在NusA-hTNFR1基础上,过表达了7种分子伴侣,筛选出tig分子伴侣对hTNFR1蛋白可溶性表达有明显的促进作用,可溶性表达量约占总量的90％;对优化后的hTNFR1表达系统的可溶性蛋白进行Ni-NTA亲和层析纯化后,TEV蛋白酶酶切去除N端的NusA标签,结合Western blot分析鉴定,获得了大量高纯度的hTNFR1蛋白。研究结果为进一步研究hTNFR1的生理学活性及其在疾病治疗方面的应用奠定了良好基础。     

一株MAs (III)脱甲基菌株的分离及功能基因的研究

[目的] 分离并鉴定三价单甲基砷（MAs （III））脱甲基菌株,对MAs （III）脱甲基菌FJ-6中arsI基因进行克隆表达,并对arsI基因表达蛋白进行功能鉴定。[方法] 利用富集培养的方法分离MAs （III）脱甲基菌株,并通过形态学、生理生化特征和16S rDNA基因进化分析进行鉴定;HPLC-ICP-MS鉴定菌株转化MAs （III）的产物为三价砷（As （III））,对菌株FJ-6的基因组进行生物信息学分析,寻找潜在的MAs （III）脱甲基酶编码基因,通过PCR扩增获得arsI全长基因,构建重组质粒pET29a-arsI,转化大肠杆菌BL21（DE3）菌株进行异源表达,通过Ni²⁺-NTA亲和层析柱纯化异源表达的蛋白,以MAs （III）为反应底物,检测MAs （III）脱甲基酶ArsI的酶学特性。通过实时定量PCR观察arsI的表达类型。[结果] Bacillus aryabhattai FJ-6在12 h内能将1 μmol/L MAs （III）完全转化为As （III）。克隆得到MAs （III）脱甲基酶表达基因arsI,构建了pET29a-arsI重组质粒并进行了表达,ArsI蛋白分子量为17.4 kDa。ArsI纯化蛋白具有较高的MAs （III）脱甲基酶的活性;荧光定量PCR实验结果表明arsI受砷诱导表达。[结论] 明确了ArsI蛋白具有MAs （III）脱甲基酶活性。     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

Trx-NAP 5融合蛋白在大肠杆菌中的表达及其活性检测

目的:用大肠杆菌表达获得重组线虫抗凝血肽5(rNAP 5),为研究开发NAP5的功能与应用提供原料来源。方法:将扩增的NAP5基因经BamHⅠ和HindⅢ双酶切后与表达载体pET-32a连接。构建好的重组表达质粒转化至大肠杆菌BL21(DE3)后,分别经IPTG和乳糖诱导表达,并探讨诱导表达条件,分析表达产物的可溶性情况。表达产物经镍亲和纯化后,用凝血酶原时间(PT)和活化部分凝血活酶时间(aPTT)检测体外抗凝活性。结果:成功构建了pET-32a/NAP5表达载体,IPTG和乳糖均能诱导目的蛋白在大肠杆菌BL21(DE3)中高效地可溶性表达。优化条件下每升LB培养基可获可溶性目的融合蛋白量达65.3mg。纯化的蛋白能明显延长PT及aPTT,7.0mg/L的蛋白平均约延长5.09倍aPTT,2.55倍PT。结论:在大肠杆菌中成功表达了具有很好生物活性的Trx-NAP5融合蛋白,为研究开发NAP5的功能与应用奠定了基础。     

Sumo分子伴侣与富含二硫键的抗真菌肽Drs基因的融合有助于其可溶性表达

利用PCR引物延伸的方法合成了分子伴侣Sumo和抗真菌肽Drosomycin的融合基因,将其插入到表达载体pET-3c中,构建出重组表达质粒pET-3c-SD,并转化至大肠杆茵BL21(DE3)中。筛选重组转化子,进行表达条件的优化和表达产物的可溶性分析。结果表明在30℃条件下,用0.5mM IPTG诱导3h 后,目的蛋白表达量最高,约占菌体总蛋白的22％,其中可溶性蛋白超过了目的蛋白的80%。经过Ni-NTA纯化后,融合蛋白的纯度可达95％以上。抑菌实验表明,该融合蛋白对白僵菌（Beauveria bassiana）具有一定的抑真菌活性。本研究证实了使用分子伴侣Sumo融合表达对具有多个二硫键的小分子多肽的表达是非常有效的。     

炭疽芽胞杆菌A16R株FtsE蛋白的可溶性表达和纯化简

目的：构建炭疽芽胞杆菌FtsE蛋白的原核表达载体,实现其在原核表达系统中的可溶性表达,并纯化融合蛋白。方法：用PCR方法从炭疽芽胞杆菌A16R株扩增得到厅sE基因片段,酶切后连接到pET28a原核表达载体,构建重组表达质粒pET28a-ftsE,转化大肠杆菌BL21（DE3）菌株,筛选可溶性诱导表达与纯化融合蛋白的条件,以获得高纯度融合蛋白。结果：构建了FtsE蛋白的融合表达载体,并在大肠杆菌中获得高效表达;在20℃下,经0．1mmol/LIPTG诱导3h表达的产物主要是可溶性蛋白,经Ni-NTA亲和层析纯化获得了高纯度的FtsE融合蛋白,经Western印迹检测,目的蛋白表达正确。结论：实现了炭疽芽胞杆菌FtsE蛋白原核表达系统的可溶性表达并获得了高纯度融合蛋白,为后续研究奠定了基础。     

血小板第四因子在原核中的高效表达、分离纯化及活性测定

目的：构建重组表达质粒pET-32c/PF4,并在原核中进行表达,以探讨其对白血病细胞系（HEL）细胞增殖的作用。方法：通过PCR方法从含有PF4基因的PQE-60/PF4质粒中扩增PF4,用NcoⅠ/HindⅢ双酶切,克隆到原核表达质粒pET-32C中,使之在BL21表达,Ni-Chelating Sepharose亲和柱纯化,用细胞集落形成方法研究重组PF4对HEL的抑制作用。结果：菌株筛选后在大肠杆菌中获得可溶性高效表达,重组PF4占菌体总蛋白的22%,肠激酶酶切除去端啧合部分,获得了高纯度的重组人血小板第四因子（rh-PF4),纯度为95%以上,活性实验发现重组PF4可抑制HEL细胞集落的形成,抑制率为47%,结论：原核表达质粒pET-32C可高效可溶性表达PF4,重组PF4对HEL细胞的增殖有抑制作用。     

T66I突变提高HIV-1整合酶可溶性研究

在研究HIV-1整合酶（IN）抗药性突变T66I时,发现这一突变同时可以提高整合酶的溶解性。原核表达了IN1–288/T66I和野生型（WT）,取菌体破碎后的上清, SDS-PAGE和his标签蛋白质染色进行分析,结果表明IN1–288/T66I可溶性约是WT的2.4倍。600 ml培养基中诱导表达IN1–288/T66I/BL21,亲和层析纯化共收获蛋白质4.72 mg。用改进的ELISA方法测定IN1–288/T66I和IN1 288/F185K /C280S链转移催化活性,结果显示两种蛋白质活性基本相当。提供了有别于F185K /C280S突变的另外一种整合酶可溶性表达的途径,IN1–288/T66I重组蛋白还可以应用到整合酶抑制剂筛选中,以获取避开T66I抗药性突变的抑制剂。     

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.