HSP70基因上游调控序列对GST基因在耻垢分枝杆菌中表达效率的 影响

将外源基因——日本血吸虫26kD抗原(Sj26GST)基因克隆 到大肠杆菌分枝杆菌穿梭质粒中,构建成四个不同的表达截体,研究它们在耻垢后分枝杆 菌(Mycobacterium smegmatis)中的表达效率。首先将含有结核杆菌热休克蛋白70(Heat Shock Protein,HSP70)的启动子的质粒pMT70用NcoI切,进行两种不同的修饰后,得到 不同的SD序列;将Sj26GST基因克隆进去。再将含HSP70启动子和Sj26GST基因的片段切下, 克隆到分枝杆菌大肠杆菌穿梭质粒pBCG2000中,筛选出不同SD序列、不同方向和不同拷 贝数的分枝杆菌表达载体四个。所表达的重组天然Sj26GST(rSj26GST)为可溶性蛋白,在SDS PAGE上分子量为26kD处可见明显的表达蛋白带。通过薄层扫描分析,发现表达质粒中双拷 贝启动子外源基因组合,表达效率最高,是单拷贝组合的16倍,占分枝杆菌菌体总蛋白 的28%。而不同的克隆方向和不同的SD序列(两者相差3个碱基)对表达效率的影响不明显。     

结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

目的：在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法：用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中,构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后,用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌,对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果：重组耻垢分枝杆菌构建成功,SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论：结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。     

胞壁表达HSP65-IL-2融合蛋白的重组耻垢分枝杆菌的构建和鉴定

目的：获得胞壁表达结核分枝杆菌热激蛋白65（HSP65）和人白细胞介素2（IL-2）融合蛋白的重组耻垢分枝杆菌。方法：将HSP65和IL-2融合基因克隆入大肠杆菌-卡介苗（E．coli-BCG）穿梭质粒pCW,构建成重组质粒HSP65-IL-2-pCW,电穿入耻垢分枝杆菌,经潮霉素抗性筛选和PCR方法选取阳性克隆;用间接免疫荧光法对重组耻垢分枝杆菌进行表型鉴定;用重组耻垢分枝杆菌免疫BALB／c小鼠,检测其诱导的抗体水平。结果：筛选获得的重组耻垢分枝杆菌增殖特性与普通耻垢分枝杆菌无明显区别;与抗人的IL-2抗体和HSP65抗体均可形成免疫印迹条带;间接荧光染色后,可见细菌表面有极强的绿色荧光;重组耻垢分枝杆菌免疫BALB／c小鼠2周后,小鼠血清中HSP65抗体平均滴度为1：4000,而生理盐水组的抗体几乎为阴性。结论：结核分枝杆菌HSP65和人IL-2融合基因在耻垢分枝杆菌胞壁获得表达,有望为结核病的预防提供有效的疫苗。     

结核杆菌eis基因的克隆及其在耻垢分枝杆菌中的表达

目的:构建结核分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物活性。方法:采用PCR技术克隆结核分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMV-eis,经酶切和测序鉴定其正确性,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc²155中,采用SDS-PAGE和Western blot检测eis基因在耻垢分枝杆菌中的表达。结果:成功构建结核杆菌eis基因穿梭表达载体pMV-eis;生长曲线说明重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE 和Western blot检测证实eis在耻垢分枝杆菌中可表达出相对分子量约42kDa的Eis蛋白。结论:成功构建了eis基因穿梭表达质粒pMV-eis,且该重组质粒在耻垢分枝杆菌中具有生物活性,为下一步研究表达产物Eis的功能奠定了一定基础。     

大肠杆菌-分枝杆菌穿梭表达质粒pBCG-2100的构建及应用

利用分子生物学方法,构建了大肠杆菌分枝杆菌(E.coliMycobacterium)穿梭表达质粒pBCG-2100,研究了编码日本血吸虫中国大陆株谷胱甘肽S转移酶(Glutathione Stransferase,GST)抗原基因在卡介苗(Bacillus Calmette Guerin,BCG)中的表达。以含人结核杆菌热休克蛋白(Heat shock protein,hsp)70基因全长序列的质粒pMT-70为模板,扩增出hsp70启动子,测序选出无错配的启动子,将其定向克隆入E.coliMycobacterium穿梭质粒pBCG-2000中,构建成E.coliMycobacterium穿梭表达质粒pBCG-2100。再将编码GST的cDNA按正确的阅读框顺序,克隆到pBCG-2100中hsp70启动子的下游,得到分枝杆菌表达质粒pBCG-GST。将pBCG-GST电转化入BCG中,筛选出重组BCG疫苗,经热诱导后所表达的重组GST(rGST)抗原,为可溶性蛋白,经纯化后,在SDS-PAGE上分子量为26kD处可见明显的表达蛋白带,其表达量占BCG菌体总蛋白的13%。Western blot提示rGST能与抗GST的抗体反应。     

结核杆菌Hsp65 与人IL-2 融合蛋白在耻垢杆菌表达

目的:构建能够分泌表达结核分枝杆菌热休克蛋白65(Hsp65)与人IL-2融合蛋白的重组耻垢分枝杆菌(recombinant Mycobacterium Smegmatis,rMs)。方法:用EcoRⅤ和HindⅢ双酶切含Hsp65-IL-2融合基因的pPRO-hsp65-IL-2载体,回收目的基因片断Hsp65-IL-2,并将其亚克隆入同样双酶切的大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22中。重组质粒pDE22-hsp65-IL-2酶切鉴定正确后,电穿孔转化MS感受态,潮霉素抗性压力筛选阳性rMs。Western-blot鉴定rMs培养上清蛋白中目的蛋白的表达。结果:重组pDE22-hsp65-IL-2质粒酶切后可获得约2000 bp片段,与预期大小一致。Western-blot结果表明,rMs培养上清蛋白中有特异性反应条带,大小为78kD,与Hsp65-IL-2融合蛋白大小相一致。结论:成功构建了大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22-hsp65-IL-2,为该rMs的免疫学特性及抗结核分枝杆菌感染的保护效果研究奠定了基础。     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的 构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法 采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌‒分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果 重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDS-PAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56 kD的Rv1776c蛋白。结论 成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

表达绿色荧光蛋白的重组耻垢分枝杆菌的构建

目的 建立可表达绿色荧光蛋白的耻垢分枝杆菌,便于对耻垢分枝杆菌进行直观检测和快速定量。方法利用PCR技术从真核表达质粒pLVTH扩增获得绿色荧光蛋白的编码基因,克隆人大肠埃希菌一分枝杆菌穿梭载体pMV261,建立重组质粒pMVGFP,并经酶切鉴定证实。利用电穿孔技术将pMVGFP转化入耻垢分枝杆菌,利用卡那霉素抗性筛选重组耻垢分枝杆菌克隆,扩大培养后直接涂片,荧光显微镜镜检。结果重组质粒pMVGFP构建正确;将重组耻垢分枝杆菌在荧光显微镜下观察,证实绿色荧光蛋白在重组耻垢分枝杆菌中的表达。结论自发释放荧光的重组耻垢分枝杆菌的成功建立,为研究结核病致病机制和快速筛选化学药物等奠定了基础。     

ESAT6-CFP10融合蛋白基因的克隆及其在耻垢分枝杆菌中的表达

目的:构建能表达ESAT6-CFP10融合蛋白的重组耻垢分枝杆菌疫苗。方法:以结核杆菌H37Rv基因组DNA为模板,克隆cfp10基因,将其插入到pGEM-T-easy载体后与esat6基因连接,构建含有esat6和cfp10融合基因的重组载体pGEM-e6c10。酶切消化pGEM-e6c10重组质粒,将esat6-cfp10基因亚克隆到pDE22分泌表达穿梭载体,以电穿孔方法将重组质粒转化至耻垢分枝杆菌。PCR筛选到的阳性克隆经温度诱导后,SDS-PAGE和Western印迹检测表达蛋白的正确性。结果:可表达出相对分子质量约23000的ESAT6-CFP10融合蛋白。Western印迹证明重组蛋白有较好的免疫原性。结论:获得能表达ESAT6-CFP10融合蛋白的重组耻垢分枝杆菌,为结核病的预防奠定了一定的基础。     

结核杆菌Hsp65与人IL-2融合蛋白在耻垢杆菌表达

目的：构建能够分泌表达结核分枝杆菌热休克蛋白65（Hsp65）与人IL-2融合蛋白的重组耻垢分枝杆菌（recombinant Mycobacterium Smegmatis,rMs）。方法：用EcoRV和HindIII双酶切含Hsp65．IL-2融合基因的pPRO-hsp65-IL-2载体,回收目的基因片断Hsp65-IL-2,并将其亚克隆入同样双酶切的大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22中。重组质粒pDE22-hsp65-IL-2酶切鉴定正确后,电穿孔转化MS感受态,潮霉素抗性压力筛选阳性rMs。Westem—blot鉴定rMs培养上清蛋白中目的蛋白的表达。结果：重组pDE22-hsp65-IL-2质粒酶切后可获得约2000bp片段,与预期大小一致。Western-blot结果表明,rMs培养上清蛋白中有特异性反应条带,大小为78kD,与Hsp65-IL-2融合蛋白大小相一致。结论：成功构建了大肠埃希菌．分枝杆菌穿梭分泌表达载体pDE22-hsp65-IL-2,为该rMs的免疫学特性及抗结核分枝杆菌感染的保护效果研究奠定了基础。     

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.