立氏立克次体外膜蛋白H基因片段的克隆表达与免疫原性分析

目的：克隆表达立氏立克次体（Rickettsia rickettsii）外膜蛋白H基因（ompH）片段并对其进行免疫原性分析。方法：采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果：获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论：pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

两种血吸虫病DNA疫苗的候选抗原基因研究

目的：以日本血吸虫基因SjFABP和SjGST原核表达产物检测二价DNA疫苗pVIVO2-SjFABP-SjGST在体内诱发的特异性抗体。方法：克隆日本血吸虫抗原基因SjFABP和sjGST,构建重组原核表达载体pET30a-SjFABP、pET30a-SjGST及真核表达载体pVIVO2-SjFABP-SjGST;将pET30a-SjFABP和pET30a-sjGST进行原核表达,并将表达产物用镍亲和柱分离纯化;采用Western印迹对日本血吸虫DNA疫苗pVIVO2-SjFABP-SjGST免疫4周后的BALB／c小鼠血清进行特异性抗体检测。结果：克隆了日本血吸虫抗原基因SjFABP（399bp）和町GST（657bp）,并构建了pET30a-SjFABP、pET30a-SjGST及pVIVO2-SjFABP-SjGST重组质粒;经Western印迹检测,pET30a-SjFABP及pET30a-SjGST原核表达的抗原蛋白均能够与经日本血吸虫二价DNA疫苗pVIVO2-SjFABP-SjGST免疫的小鼠的血清产生特异性免疫反应。结论：日本血吸虫町尉即和町GST基因的原核表达系统成功建立;原核表达的抗原蛋白具有免疫原性;以原核表达产物可检测日本血吸虫DNA疫苗pVIVO2-SiFABP-SiGST在体内诱发的特异性抗体。     

沙门菌毒力基因spvB的原核表达及其多克隆抗体的制备

目的:构建沙门菌毒力基因spvB的原核表达载体,诱导表达纯化SpvB蛋白并以其为抗原免疫小鼠,制备多克隆抗体。方法:利用生物信息学软件对SpvB进行分析,选取抗原性较高、易表达的氨基酸序列作为克隆序列,以携带spvB基因的鼠伤寒沙门菌为模板,PCR扩增目的片段后与原核表达载体pET28a(+)连接;将质粒pET28a-SpvB转化大肠埃希菌BL21(DE3)后诱导表达并纯化。目的蛋白免疫小鼠,制备抗SpvB多克隆抗体,Western blot检测抗体特异性。结果:成功构建spvB原核表达载体,经IPTG诱导结果显示,重组蛋白表达且主要存在于包涵体中,将纯化后的蛋白免疫小鼠Western blot检测血清中抗体与SpvB特异性结合。结论:获得具有免疫原性的SpvB蛋白及其多克隆抗体,为进一步研究该基因的功能奠定基础。     

融合蛋白CBD-SPA“Z”基因的构建、表达与活性鉴定

针对金黄色葡萄球菌蛋白A(SPA)层析柱的应用问题,将SPA的Z结构域基因与纤维素结合结构域(CBD)重组,并在Z结构域C端引入1个半胱氨酸(Cys),构建并表达出一种新型免疫亲和材料。利用基因工程技术将质粒pEZZ18中的Z基因插入到含有CBD的质粒pET35b(+)质粒中,构建出原核表达载体pET35b(+)-ZCys,经IPTG诱导表达,获得CBD-Protein Z融合表达蛋白。pET35b(+)-Z-Cys在E.coli BL21中正确表达,所表达的融合蛋白具有与哺乳动物IgG抗体结合的生物学活性和与纤维素结合的活性。结果证明CBD-Protein Z蛋白具有Z结构域与CBD结构域的活性,预计能在免疫亲和层析中用于IgG抗体的纯化。     

鸭坦布苏病毒E蛋白的原核表达及多克隆抗体的制备

目的用RT-PCR技术扩增鸭坦布苏病毒(DTMUV)AH-F10株E基因,并克隆至pET32a(+)载体,构建重组表达质粒pET32a-E,表达E蛋白。方法重组表达质粒转化感受态细胞BL21(DE3),经IPTG诱导后,获得含6个His标签的融合蛋白,大小约54kDa。表达的蛋白以包涵体形式存在。对目的蛋白进行纯化,用纯化的E蛋白免疫Balb/c小鼠,制备多克隆抗体血清。结果SDS-PAGE和Western blot试验结果表明E基因在大肠埃希菌中成功表达,并能与抗DTMUV多克隆抗体产生特异性反应,具有良好的反应原性。间接免疫荧光试验表明免疫小鼠后获得的多克隆抗体能与DTMUV反应。结论本研究为DTMUV新型疫苗和诊断试剂盒的进一步研究奠定了基础。     

人乳头瘤病毒58型E6E7融合蛋白的原核表达、纯化及鉴定

目的:构建pET-42a(+)-HPV58E6E7原核表达质粒,诱导表达人乳头瘤病毒(HPV)58型E6E7融合蛋白。方法:采用PCR方法扩增出HPV58 E6E7融合基因的全长序列,利用DNA重组技术将其定向插入原核表达载体pET-42a(+)中,构建pET-42a(+)-HPV58E6E7原核表达质粒,用限制性内切酶酶切和核酸序列检测对重组质粒进行鉴定;将其转入宿主菌大肠杆菌BL21进行诱导以表达HPV58E6E7融合蛋白,并用谷胱甘肽琼脂糖树脂纯化回收HPV58E6E7融合蛋白,用SDS-PAGE及Western印迹鉴定表达蛋白的相对分子质量及抗原性。结果:PCR、限制性内切酶酶切和核酸序列检测证实重组质粒中插入的目的基因大小、方向正确;HPV58E6E7融合蛋白得到高效原核表达及纯化,表达蛋白的分子大小正确,抗原性良好。结论:pET-42a(+)-HPV58E6E7原核表达质粒构建成功,HPV58E6E7融合蛋白得到高效表达及有效纯化,为检测HPV58型治疗性疫苗的免疫效果提供了抗原。     

重组血型抗原结合黏附素的免疫原性初步探讨

目的 构建幽门螺杆菌(Helicobacter pylori, Hp)血型抗原结合黏附素(blood group antigen-binding adhesin,babA)基因,表达纯化重组BabA蛋白;初步探索口服免疫BabA配伍双突变大肠埃希菌(E.coli)热不稳定毒素(double mutant heat-labile toxin, dmLT)佐剂在BALB/c小鼠中的免疫保护作用。方法 从Hp SS1株基因组DNA中扩增babA编码基因,构建原核表达质粒pET28a-babA,转化入E.coli BL21(DE3)感受态细胞,IPTG诱导表达,His标签纯化。将纯化的BabA蛋白配伍dmLT免疫佐剂口服免疫BALB/c小鼠,生理盐水对照;末次免疫后2周小鼠眼眶静脉采血分离血清,分析特异性体液免疫应答;然后进行Hp胃部活菌感染,感染27周采集脾淋巴细胞进行细胞免疫分析并对小鼠胃部细菌定植量进行检测。结果 重组质粒pET28a-babA基因序列正确;重组蛋白BabA以包涵体形式表达,相对分子质量56 000,纯度为95.9%;重组BabA蛋白配伍dmLT佐剂口服免疫小鼠可诱导产生...     

人IL-6基因的克隆及其在原核生物中表达及条件的优化

构建人白细胞介素6(IL-6)的原核表达载体并优化其表达条件,为IL-6的高效表达提供试验依据。以人T细胞cDNA为模板通过PCR方法扩增IL-6基因,将其克隆到原核表达载体pET28a(+)中,酶切及测序鉴定重组体。将构建好的重组质粒转化大肠杆菌BL21(DE3),用IPTG进行诱导表达,产物用Western blotting及人IL-6检测试剂盒分析鉴定。在保持菌种不改变的前提下,分别改变IPTG的浓度、培养时间、卡那霉素浓度、培养温度等来优化IL-6表达条件。结果显示,原核表达载体pET28 a(+)-IL-6成功构建,可在大肠杆菌BL21(DE3)中诱导表达,得到相对分子质量约22 kD的IL-6蛋白,经Western blotting鉴定正确,经人IL-6试剂盒检测显示具有较高的免疫活性。在IPTG浓度400μmol/mL,卡那霉素浓度50μg/mL,40℃培养6 h的条件下,目的蛋白表达量最高,可占总蛋白表达量的40%。成功构建人IL-6原核表达载体且获高效表达,为研究IL-6生物学活性及产品开发提供了试验基础。     

流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A融合蛋白的表达及其免疫原性研究

本研究构建了表达甲型流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A(PEA)融合蛋白的原核表达载体,根据铜绿假单胞菌外毒素A(PEA)核苷酸序列设计突变PCR引物并实施突变PCR,以获得PEA基因编码区第553位氨基酸密码子缺失的突变PEA(ntPE),从而产生无毒性的PEA突变基因,然后用合成的M2e编码区替换ntPE基因中的非必需区Ib,产生ntPE-M2e嵌合基因。将该嵌合基因导入pET表达载体以构建原核表达载体,将表达产物胶回收后与弗氏不完全佐剂联合皮下免疫BALB/c小鼠,终免两周后用5个LD50流感病毒A/PR/34/8株进行攻击。取动物血清作ELISA并取脾脏作ELISPOT试验结果表明,免疫组可以诱导小鼠产生抗M2e特异性抗体反应和细胞免疫反应并能够抑制病毒在肺内的复制。本研究为甲型流感病毒广谱疫苗的进一步研发打下了基础。     

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.