幽门螺杆菌重组vacA-CtxB蛋白的原核表达及免疫学研究

目的原核表达幽门螺杆菌pQE-vctB(含H.pylori细胞空泡毒素毒性片段与霍乱毒素B亚单位的融合基因)在E.coil DH5α中诱导表达,以获得重组蛋白,鉴定其抗原性和免疫原性,为制备防治H.pylori感染的口服疫苗奠定基础。方法 (1)pQE-vctB转化E.coli DH5α,IPTG诱导表达重组蛋白VCTB,Western blot分析抗原性,镍离子柱纯化。(2)重组蛋白VCTB口服免疫小鼠,ELISA检测小鼠血清特异性IgG、小肠冲洗液IgA,以鉴定其免疫原性。结果重组蛋白VCTB经SDS-PAGE分析相对分子量(Mr)约为40kD,亲和层析后可获得纯度为92%以上的蛋白。Western blot分析显示能与VacA抗血清反应。ELISA检测显示,免疫小鼠的血清特异性抗体IgG,肠粘液IgA显著高于VacA对照组(P0.01)。结论 vacA和ctxB融合基因原核表达质粒转化E.coli DH5α表达菌获得了重组蛋白VCTB,表达量较高,纯度较高,有良好的抗原性和免疫原性,口服免疫小鼠可明显提高其免疫效果,产生较高水平的IgA,可用于制备防治H.pylori感染的口服疫苗。     

幽门螺杆菌VacA重组蛋白表达、纯化及鉴定

目的研究幽门螺杆菌空泡毒素（VacA）编码基因在大肠埃希菌中的表达及纯化重组蛋白的抗原性。方法将PET32a-vacA-E.coli BE21（DE3）工程菌株常规培养,碱裂解法小量提取重组质粒DNA,琼脂糖凝胶电泳进行酶切鉴定,基因测序法进行插入基因序列分析。重组蛋白采用IPTG诱导表达,镍亲和层析原理提纯,ELISA法检测其抗原性。结果经酶切鉴定表明,插入的基因片段全长约2240bp,测序分析及与Genebank比较,可以肯定插入片段为vacA基因,ELISA法检测重组蛋白具有良好的抗原性。结论VacA重组蛋白在大肠埃希菌中成功表达,重组蛋白具有良好的抗原性。     

His-FemA融合蛋白表达载体的构建及其在原核细胞中的表达

目的 构建His标签的金黄色葡萄球菌甲氧西林耐药相关蛋白FemA的融合蛋白表达载体,并在大肠埃希菌中表达,为进一步研究femA基因的生物学功能和临床应用奠定基础.方法 根据GenBank中金黄色葡萄球菌femA基因序列,利用Primer Premier 5.0设计PCR引物,并在引物的5'加入BamHI及SalI酶切位点;以金黄色葡萄球菌基因组DNA为模版,PCR扩增出femA基因片段.将目的DNA片段及质粒pQE30分别进行双酶切、连接并转化大肠埃希菌DH5α;阳性克隆以PCR、双酶切及测序进行鉴定.将鉴定正确的pQE30-femA重组质粒转化大肠埃希菌JM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-femA融合蛋白;采用SDS-PAGE及Western blot分析对表达蛋白进行验证.结果 经PCR、双酶切签定及序列测定,证实重组质粒pQE30-femA构建成功;重组质粒pQE30-femA转化大肠埃希菌JM109经IPTG诱导后,SDS-PAGE和Western blot分析显示表达出53 kD目的蛋白;经Bandscan软件分析,目的蛋白质在4h的表达量占细胞总蛋白的27.5％.结论 成功构建了His-FemA原核表达载体(pQE30-femA),并在大肠埃希菌中高效表达.     

肠产毒性大肠埃希菌F4ac菌毛蛋白FaeG亚单位的原核表达及免疫学鉴定

目的克隆并表达肠产毒性大肠埃希菌（ETEC）F4ac菌毛蛋白亚单位FaeG,为制备预防幼畜ETEC感染的疫苗奠定基础。方法以ETEC（C83902）基因组DNA为模板,PCR扩增faeG基因,插入原核表达质粒pGEX-6P-1,构建重组质粒pGEX-faeG。将pGEX-faeG转化大肠埃希菌BL-21I,PTG诱导表达。SDS-PAGE分析表达蛋白的相对分子质量和表达形式,Western blot鉴定其抗原性。将表达菌超声破碎后离心提取包涵体制备抗原,经口灌喂免疫小鼠,检测小鼠血清中抗FaeG的IgGI、gA,鉴定其免疫原性。结果扩增的faeG基因全长786 bp,与基因文库中的faeG基因同源性达96%。重组质粒pGEX-faeG经PCR及双酶切鉴定确有插入片段,且序列完整。表达产物FaeG相对分子质量约53 kD,主要存在于碎菌后的沉淀中,以包涵体形式表达。Western blot显示该蛋白可与ETEC F4ac阳性血清特异性结合,免疫后小鼠血清抗FaeG IgGI、gA明显高于PBS和GST对照组。结论成功构建了ETEC F4ac菌毛蛋白亚单位FaeG的重组质粒pGEX-faeC,表达了重组蛋白FaeG,该蛋白具有良好的抗原表达了重组蛋白FaeG,该蛋白具有良好的抗原性和免疫原性,可用于研制预防幼畜ETEC感染的疫苗。     

CTX-M-14型超广谱β-内酰胺酶的序列分析与原核表达

目的对大肠埃希菌所产CTX-M-14型超广谱β-内酰胺酶（ESBLs）进行基因克隆和重组表达,探讨其特性。方法以产CTX-M-14型超广谱β-内酰胺酶大肠埃希菌12号菌总基因组DNA为模板,PCR扩增CTX-M-14,将其克隆入pUCm-T Vector载体后测定该核苷酸序列;再将基因编码区克隆到原核表达载体pET-28α,构建含CTX-M-14基因的重组表达质粒,转化到大肠埃希菌BL21中进行IPTG诱导表达。SDS-PAGE电泳鉴定表达的酶蛋白后再过Ni-NTA柱纯化。结果PCR扩增出大小为876bp的基因片段,与GenBank上同类酶的基因序列同源性为100％。大肠埃希菌BL21转化pET-28a／CTX-M-14重组质粒后,ESBLs试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示蛋白分子质量大约为30KD。结论成功表达重组的CTX-M-14型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定基础。     

幽门螺杆菌napA基因在乳酸菌中的表达及免疫原性分析

本实验目的是在乳酸菌中表达幽门螺杆菌（Helicobacter pylori,H.pylori） 中性粒细胞激活蛋白(NAP),口服免疫小鼠后检测其免疫原性。在实验中利用了分子克隆技术构建携带nap基因的重组原核表达质粒pNICE:sec-nap,将重组质粒转化乳酸菌NZ9000株,筛选鉴定阳性菌落,诱导表达的NAP蛋白用SDS-PAGE和Western blot进行鉴定。将雌性ICR（CV级）小鼠随机分为4组,分别用PBS、携带空质粒的乳酸菌、携带pNICE:sec-nap的乳酸菌、灭活的H.pylori 灌胃。免疫7次后检测其特异性IgG和IgA的产生。成功扩增了nap基因并构建了重组原核表达质粒pNICE:sec-nap,转化乳酸菌NZ9000后经nisin诱导可表达Mr约17kDa的重组蛋白,表达量约占菌体总蛋白量的9.5%,表达的蛋白能与兔抗H.pylori 血清特异性反应,具有良好的免疫原性。携带pNICE:sec-nap质粒的乳酸菌刺激产生的IgG水平明显高于携带空质粒组,与灭活H.pylori组没有明显的差异,但其刺激产生的IgA水平明显高于其他组。以上结果说明表达NAP蛋白的乳酸菌口服免疫小鼠后,能够刺激小鼠产生特异的IgG和IgA,对幽门螺杆菌疫苗的研究开发具有理论的意义。为乳酸菌作为抗原递送载体的研究和H.pylori口服疫苗的开发提供一定的实验基础。     

肺炎支原体双蛋白多抗原表位表达载体的构建

目的构建肺炎支原体(Mp)双蛋白多特异抗原表位表达载体,提高重组蛋白抗原的敏感性。方法应用生物信息学方法筛选Mp P116粘附蛋白抗原表位序列,PCR点突变技术获取P116蛋白基因片段,与pMD-T载体重组,转入大肠埃希菌JM109,通过限制性酶切图谱和基因序列分析鉴定重组质粒。酶切回收P116基因片段与pGEX 6P-1-P1 DNA重组,转入大肠埃希菌JM109菌株。用Glutathione Sepharose 4B纯化重组蛋白,SDS-PAGE分析表达产物的相对分子量,用Mp免疫血清进行免疫印迹试验,鉴定重组蛋白的免疫原性。结果 PCR点突变扩增Mp黏附蛋白P116的基因片段为597 bp,该基因片段与已知的基因库序列分析比较,除两个突变位点由UAG突变为UGG外,其余核苷酸序列同源性为100%。SDS-PAGE分析多表位重组蛋白相对分子质量(Mr)为77.8 kDa。免疫印迹结果显示,Mp兔多价血清能与纯化的78KDa的重组蛋白发生免疫反应。结论本研究成功构建了Mp双蛋白多表位的表达载体。该表达载体表达的重组蛋白具有Mp特异的免疫反应性。重组蛋白的敏感性有待进一步鉴定。     

腐败梭菌α毒素基因的克隆表达及其类毒素的免疫原性研究

根据GenBank公布的腐败梭菌α毒素基因序列,设计了一对引物,并以腐败梭菌基因组为模板,经PCR特异性扩增出腐败梭菌菌株HeB01的α毒素基因。序列分析表明,该基因产物大小为1323bp,与GenBank报道的4个腐败梭菌参考菌株α毒素基因序列同源性高于99·5%。将扩增的α毒素基因定向克隆到原核表达载体pQE30中,得到重组质粒pQE30-α,将重组质粒转化大肠杆菌M15中,得到重组菌株M15(pQE30-α),经IPTG诱导后,SDS-PAGE分析可见表达的48kD特异条带;Westernblot和ELISA检测实验表明:表达的α毒素与抗天然α毒素抗体发生特异性反应,说明α毒素蛋白具有较好的免疫原性。将表达的α毒素蛋白制成类毒素疫苗,免疫小鼠后,具有一定的保护能力,表明该重组菌株有望作为腐败梭菌基因工程类毒素疫苗的候选生产菌株。     

幽门螺杆菌VacA N端在巨噬细胞中表达对其细胞因子 分泌功能的影响

构建pDsRed-Monomer-C1/vacA N端真核表达载体,研究幽门螺杆菌空泡毒素单一毒力决定簇对THP-1巨噬细胞细胞因子分泌的影响.PCR扩增vacA目的基因片段,克隆人真核表达载体pDsRed-Monomer-C1中,经酶切、PCR及测序鉴定后,转染THP-1巨噬细胞中,Western blot和荧光显微镜鉴定VacA蛋白在细胞中的表达;电子显微镜和中性红摄人法观察巨噬细胞的空泡样变;ELISA法检测巨噬细胞培养上清TNF-α、IL-1β含量.重组质粒转染THP-1巨噬细胞24h,部分细胞胞浆中出现大小不等的空泡,且重组质粒组培养上清中TNF-α、IL-1β含量明显高于空质粒组和阴性对照组(P＜0.001),二硫代氨基甲酸吡咯烷(pyrrolidine dithiocarbamate,PDTC)下调细胞因子的分泌.结果显示,成功构建pDsRed-Monomer-C1-vacA真核表达载体;VacA蛋白瞬时高表达上调THP-1巨噬细胞分泌TNF-α、IL-1β;核因子kB(nuclear factor kappaB,NF-kB)可能参与调节VacA诱导的THP-1巨噬细胞的分泌.     

结核分枝杆菌19kD-ESAT6融合蛋白的克隆表达及纯化

运用聚合酶链反应( PCR) 技术从结核分枝杆菌标准株H37Rv 中扩增获得19kD 脂蛋白和esat-6 基因片段, 将目的片段分别克隆到pMD18-T 载体, 再亚克隆目的片段到同一表达载体pET-21a, 构建重组表达载体pET21a-19kD-ESAT6, 转化至大肠埃希菌BL21( DE3) , 表达纯化后用蛋白质印迹法( Western blot) 分析其抗原反应性。结果成功构建了重组质粒pET21a-19kD-ESAT6, 并在大肠埃希菌中获得高效表达。SDS-PAGE 结果显示, 在相对分子质量( Mr) 约29 ×103 处有表达条带。estern blot 结果表明, 重组蛋白19kD-ESAT6 与确诊的结核病患者血清发生特异性免疫反应, 具有特异的抗原性。本研究构建的结核分枝杆菌19kD-ESAT6 融合蛋白为结核病血清学诊断试剂的开发提供了依据。     

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.