水稻非特异性脂质转移蛋白的原核表达、纯化及抑菌功能

将编码水稻非特异性脂质转移蛋白 (nonspecificlipidtransferprotein ,nsLTP)基因 (LTP110 )的克隆到硫氧还蛋白融合表达载体PET32a( )中 ,在BL2 1(DE3)trxB-宿主菌中实现了融合蛋白的高表达。通过Ni2 chelatingSepharosefastflow柱纯化融合蛋白后 ,通过肠激酶酶切再过该亲和柱得到了重组LTP110。CD谱扫描表明重组蛋白质与体内提取的nsLTP二级结构相似 ;荧光脂质结合实验表明该蛋白质具有结合脂肪酸分子的活性。对该蛋白质的抑菌功能进行研究后表明 ,LTP110具有抑制稻瘟病菌孢子萌发的功能 ,在较低浓度即能发挥活性     

家蝇幼虫抗菌肽Attacin基因的克隆表达及抑菌生物学活性

目的克隆家蝇幼虫Attacin抗菌肽基因．构建原核融合表达载体,建立Attacin体内抗菌活性检测系统,优化表达和纯化Attacin目的蛋白,并初步研究其抗菌生物学功能。方法以pUC m-T／Attacin重组质粒为模板,设计特异性引物,PCR扩增Attacin编码区序列,分别克隆至原核表达载体pET30a（＋）和pGEX-4T-1。构建原核重组质粒,转化大肠埃希菌,表达重组Attacin蛋白,并在大肠埃希菌中体内检测Attacin的抗菌活性。利用亲和层析柱纯化重组融合蛋白Attacin,SDS-PAGE进行纯度分析,琼脂糖平板抑菌试验鉴定其生物活性。结果pET30a（a＋）／Attacin和pGEX-4T—1／Attacin重组质粒分别转化大肠埃希菌后,以IPTG诱导表达,与未诱导对照相比,含有重组质粒的宿主菌生长受到抑制。从pET30a（＋）／Attacin重组质粒的表达宿主菌中未能获得His-Attacin融合蛋白,而从pGEX-4T—1／Attacin重组质粒转化菌种获得GST-Attacin融合蛋白。SDS-PAGE分析表明Attacin重组蛋白分子量与预期结果一致,琼脂糖平板抑菌试验显示重组Attacin具有抗菌活性。结论Attacin基因在原核系统中成功表达,并且纯化后具有抑菌活性,为下一步研究Attacin的生物学功能及其应用开发奠定了基础。     

痢疾志贺菌侵袭素IpaC的原核表达及纯化复性

目的为进一步研究痢疾志贺菌侵袭素IpaC蛋白的侵袭活性,必须先获得IpaC重组融合蛋白。方法将IpaC基因亚克隆至表达载体pET-24α(+)中,转化至E.coli BL21(DE3)表达宿主菌中,采用IPTG优化诱导表达,然后纯化复性重组蛋白。结果重组蛋白最佳表达条件为0.50mmol/L IPTG、30℃诱导6h,获得分子量约为33kDa的IpaC重组蛋白。Western blotting证实了重组蛋白的特异性。IpaC重组蛋白主要以包涵体形式在宿主菌中表达,通过纯化后得到单一的目的蛋白。结论成功构建IpaC原核表达体系,并获得了蛋白的高效表达及优化。初步建立了IpaC重组蛋白的纯化方案。为进一步研究IpaC的侵袭性作用奠定了基础。     

人丝氨酸蛋白酶抑制因子Hespintor的基因克隆与序列分析

目的:克隆得到人丝氨酸蛋白酶抑制因子Hespintor基因,并应用生物信息学方法进行序列分析。方法:提取HepG2细胞总RNA,RT-PCR扩增Hespintor基因片段,连接至pMD 20-T克隆载体中,转化JM109宿主菌,蓝白斑法筛选阳性克隆菌落,菌落PCR及测序鉴定。利用在线工具软件对Hespintor进行信号肽预测、亚细胞定位、结构域、三级结构、基因染色体定位及组织分布表达分析。结果:人丝氨酸蛋白酶抑制因子Hespintor基因全长285bp,共编码94aa,其中1-23aa编码信号肽,符合亚细胞定位于细胞外的预测;53-93aa编码一个典型的Kazal结构域。Hespintor基因的染色体定位于5号染色体短臂3区3带1亚带(5q33.1);正常组织中仅在睾丸有表达,而肿瘤组织中仅在生殖细胞瘤有表达。结论:Hespintor是一个在机体中静止表达的Ka-zal型丝氨酸蛋白酶抑制因子家族的分泌型新成员。     

蛇毒锯鳞蝰素融合蛋白的发酵与纯化

研究大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合蛋白的发酵和纯化工艺。将Ecs基因插入表达载体pTXB1,转化E.coliBL21(DE3)构建工程菌。对工程菌进行补料分批培养并诱导表达,研究培养基、培养和诱导时间对工程菌生长和目的蛋白表达的影响,几丁质亲和层析纯化Ecs融合蛋白,经DTT裂解后,检测Ecs活性。发酵后菌体湿重可达75g/L,融合蛋白表达量约占总蛋白的35%,重组质粒在BL21宿主菌中传代稳定。亲和层析纯化后,得到Ecs单体,得率为28mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。优化了Ecs融合基因工程菌的发酵和纯化条件,为规模化生产奠定基础。     

骨桥蛋白RGD黏附序列多拷贝短肽的表达、纯化及生物学活性测定

目的:用大肠杆菌表达骨桥蛋白RGD黏附序列6拷贝短肽,经分离纯化后检测其生物学活性.方法:运用基因重组技术,将骨桥蛋白RGD黏附序列的核酸片段首尾相连,与携带GST编码序列的原核表达载体连接构建融合蛋白表达质粒pGEX-3X-RGD.将重组质粒转化宿主菌后,对诱导融合蛋白表达的条件进行优化.表达产物GST-RGD经谷胱甘肽-亲和层析纯化后,分别检测其对骨桥蛋白诱导的血管平滑肌细胞黏附和迁移的影响.结果:所构建的含有6个拷贝短肽的GST-RGD融合蛋白可在大肠杆菌中以包含体的形式进行表达.用十二烷基肌氨酸钠变性溶解包含体及透析复性后,经亲和层析可得到高纯度的GST-RGD(6)融合蛋白.GST-RGD(6)融合蛋白能特异性的抑制骨桥蛋白诱导的血管平滑肌细胞的黏附和迁移.结论:骨桥蛋白RGD黏附序列6拷贝短肽可在大肠杆菌中高效表达,纯化的GST-RGD融合蛋白具有抑制血管平滑肌细胞黏附和迁移的活性.     

乙型肝炎病毒DNA聚合酶反式调节蛋白HBVDNAPTP1的表达、纯化与初步鉴定

目的构建HBVDNAPTP1基因的原核表达载体,诱导其在大肠埃希菌中表达,并对融合蛋白进行纯化。方法利用逆转录-PCR获得乙型肝炎病毒(HBV)DNA聚合酶(Polymerase)反式调节人类新基因HBVD-NAPTP1,测序正确后插入至原核表达载体pET-32a(+)中,转化BL21(DE3)宿主菌进行诱导,并利用组氨酸亲和层析方法对融合蛋白进行纯化。结果 HBVDNAPTP1原核表达载体转化宿主菌后,经0.5 mmol/L IPTG、30℃诱导5 h获得了分子量约为31 kD的HBVDNAPTP1融合蛋白的优化表达,Western blotting证实融合蛋白的特异性。亲和层析纯化后得到较纯的HBVDNAPTP1融合蛋白,每升培养菌液中可获得2.24 mg的纯化蛋白。结论成功获得纯化的HBVDNAPTP1融合蛋白,为今后开展HBVDNAPTP1的生物学功能研究奠定了物质基础。     

水稻osRACB基因的原核表达及其蛋白质产物的生化特性鉴定

Rac蛋白作为高等植物中已知的惟一一类分布广泛的信号GTP结合蛋白 ,在植物体众多生命活动调节中起着分子开关的作用。实验将水稻Rac家族新成员osRACB基因克隆于原核表达载体pET 2 8a中 ,转化大肠杆菌BL2 1(DE3)宿主菌 ,经IPTG诱导实现了目标融合蛋白质的高效表达。通过Ni2 NTA柱纯化 ,获得纯化的目标融合蛋白质 ,经凝血酶作用后得到osRACB非融合蛋白质。该蛋白质经谷胱甘肽氧化还原体系复性和超滤浓缩后 ,用于体外功能鉴定。结果显示 ,osRACB蛋白具有与GTP特异性结合的活性以及水解GTP的功能。与另一Rac蛋白osRACD相比较 ,osRACB具有更强的GTP结合活性和较弱的GTP水解活性。     

还原酶缺陷型大肠杆菌对重组蛋白溶解性的影响

探讨大肠杆菌细胞质氧化还原环境对重组蛋白溶解性的影响。选择含有1对二硫键的牛碱性成纤维细胞生长因子（BbFGF）作为简单蛋白的模式分子,选择含有2对二硫键的人抗HBsAg单链抗体（HBscFv）作为复杂蛋白的模式分子,分别构建表达质粒并转化普通宿主菌和还原酶缺陷型宿主菌E. coli Origami(DE3),比较表达产物的溶解性和纯化产物的活性。结果发现,BbFGF在普通宿主菌中大部分形成包涵体,在Origami(DE3)中为可溶性表达,但表达量降低。两种工程菌的表达产物经离子交换和肝素亲和层析两步纯化后,MTT法测定活性,发现来自还原酶缺陷型宿主菌的BbFGF活性高于普通宿主菌表达产物,二者的ED₅₀分别是1.6 ng/mL和2.2 ng/mL;HBscFv在两种宿主菌中均形成包涵体,包涵体以6 mol/L盐酸胍缓冲液溶解后,镍离子螯合亲和层析纯化并透析复性,间接ELISA测定抗原结合活性,发现二者活性无明显差异,但在Origami(DE3)菌体破碎后的的上清中可检测到HBscFv活性,纯化后产量为1～2 mg/L,而在普通宿主菌破碎后的上清中检测不到HBscFv活性。上述结果说明,改变宿主菌细胞质氧化还原环境对于含有1～2对二硫键的重组蛋白的可溶性表达具有明显促进作用。     

粪肠球菌Ace重组蛋白的表达、纯化及黏附活性研究

构建含粪肠球菌表面蛋白Ace保守序列A的重组载体,在大肠埃希菌M15中进行诱导表达、纯化表达产物,研究其黏附活性。以粪肠球菌标准株JH2-2为模板进行PCR扩增ace基因保守序列A,构建重组质粒pQE30/Ace,转化至表达宿主菌M15中进行诱导表达,利用SDS-PAGE和Western blot进行分析和鉴定表达结果,Ni-NTA亲和层析柱纯化重组蛋白,同时用纯化的Ace重组蛋白免疫新西兰兔,用所得相应的抗血清分别进行黏附和黏附抑制实验。结果可见,构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片段为ace基因保守序列A,测序结果与Genbank上登录序列完全一致;SDS-PAGE分析显示,重组工程菌表达了一相对分子质量(Mr)约为37 ku的目的蛋白条带,Western blot检测其能与6×His单克隆抗体发生特异性反应。粪肠球菌JH2-2能够黏附于胶原蛋白Ⅰ表面;抗Ace多克隆抗体可抑制46℃培养的JH2-2对胶原蛋白Ⅰ的黏附,这种抑制作用与其稀释度呈负相关。构建的原核表达载体PQE30/Ace在E.coli M15中成功地表达,纯化的重组Ace蛋白具有胶原黏附活性。     

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.