肺炎链球菌体内启动子诱捕文库的构建与初步分析

构建肺炎链球菌体内启动子诱捕文库(promoter-trap library),用于筛选肺炎链球菌体内诱导的基因。以穿梭质粒pEVP3为骨架,将无启动子的galU基因作为体内报告基因定向克隆到pEVP3上,与无启动子的体外报告基因lacZ基因融合,构建用于筛选体内诱导基因的载体pEVP3-galU;再把肺炎链球菌基因组DNA的随机酶切片段(200~500bp)克隆到此载体galU基因上游的Bgl II位点,构建了启动子诱捕文库,并转化肺炎链球菌galU缺陷菌株,获得相应的菌株库。文库大约覆盖基因组全长的5倍,插入率达到90%以上,保持了较高的复杂性。对得到的约450 000个肺炎链球菌转化子进行体内、外初步分析,那些从小鼠体内分离出的细菌,并在X-gal平板上为白色的菌落表明galU报告基因上游含仅在体内表达的启动子,提示所构建的启动子诱捕文库可用于筛选肺炎链球菌的体内诱导基因。     

痘苗病毒增强子样序列的筛选及应用研究

目的从痘苗病毒基因组中筛选原核增强子样序列，构建携带原核增强子样序列的表达载体，探讨其对干扰素基因表达的影响。方法采用氯霉素乙酰转移酶基因（cat）作为报告基因，从痘苗病毒基因组中筛选具有原核增强子样活性的序列，构建携带增强子样序列VV1的表达载体，表达干扰素基因和检测干扰素活性。结果从痘苗病毒天坛株DNA基因组中筛选出18个在大肠埃希菌中具有增强活性的序列，从中筛选到两个原核增强子样序列VV1和VV16，VV1正反向分别可使lacZ基因活性提高10．9倍和3．8倍，VV16正反向分别可使lacZ基因活性提高9．0倍和4．1倍；证实它们的增强活性体现在转录水平；用痘苗病毒增强子样序列VV1构建的表达载体，其表达的IFN—a2b型干扰素比原表达载体活性高2．6倍。结论从痘苗病毒天坛株DNA基因组中筛选到2个原核增强子样序列一携带有痘苗病毒增强子样序列的表达载体可提高干扰素基因的表达水平。     

利用IVIAT技术初步筛选伤寒沙门菌的体内诱导基因

目的 利用体内诱生抗原筛选技术初步筛选伤寒沙门菌的体内诱导基因.方法 首先将3个伤寒病人恢复期的血清等体积混合,用体外培养的伤寒沙门菌全菌体、超声裂解产物及加热变性裂解产物充分吸收处理.然后采用pET-30a/b/c表达载体系统构建伤寒沙门菌Ty2菌株的基因组表达文库,并以吸收后的血清为探针,筛选伤寒沙门菌基因组表达文库.结果 从5 000个克隆中获得5个阳性克隆,对阳性克隆测序和序列检索分析表明,涉及 6 个开放阅读框.结论 本实验利用IVIAT筛选体内诱导基因的各个程序是正确的,并初步筛选到伤寒沙门菌的体内诱导基因.     

原核增强子样序列的筛选及其应用研究

目的 从大肠埃希菌C600株染色体基因组中筛选原核增强子样序列,构建携带原核增强子样序列的表达载体,探讨其对干扰素基因表达的影响.方法 采用氯霉素乙酰转移酶基因（CAT）作为报告基因,从大肠埃希菌C600株染色体基因组中筛选具有原核增强子样活性的序列,构建携带增强子样序列的表达载体,表达干扰素基因和检测干扰素活性.结果 从大肠埃希菌C600株染色体基因组中筛选到一个原核增强子样序列3A,其正、反向增强活性分别能提高β-半乳糖苷酶活性7.11和2.93倍.证实它的增强活性体现在转录水平;用原核增强子样序列3A的功能区3P3构建的表达载体,其表达的IFN-α2b型干扰素比原表达载体活性高3.7倍.结论 从大肠埃希菌C600株染色体基因组中筛选到一个原核增强子样序列3A,携带有原核增强子样序列的表达载体可提高干扰素基因的表达水平.     

MRSA耐药相关TG-TPase嵌合基因的原核表达与纯化

目的 设计、构建耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcus aureus,MRSA)耐药相关TGTPase嵌合基因,进行原核表达、纯化探索,为进一步利用其酶学活性建立抑制剂筛选系统奠定基础.方法 通过引物设计,从盐平板法筛选的MRSA菌株中分别克隆PBP2的TG基因片段和PBP2a的TP片段,分别克隆入T载体,对阳性重组子进行酶切/再连接,构建TG-TPase嵌合基因,经核苷酸测序鉴定正确的嵌合基因再亚克隆到pET22b,构建表达载体,转化Rosetta(DE3)plysS,用LPTG进行诱导表达,并对表达的蛋白进行SDS-PAGE、质谱和Westem blotting鉴定.小量发酵重组菌,对嵌合基因表达产物进行初步纯化.结果 从13株临床分离的金葡菌中筛选出2株高耐药性MRSA菌株,用PCR法从中成功地克隆到青霉素结合蛋白PBP2的TG片段和PBP2a的TP片段,构建了TG-TPase嵌合基因及其原核表达载体,并在大肠埃希菌中表达,产量达菌体总蛋白的43%.融合蛋白纯化分析表明,嵌合蛋白以包涵体形式存在,在变性条件下经Ni-NTA亲和柱纯化,纯度达90%以上.结论 成功设计并构建了耐药性金葡菌TG-TPase嵌合基因及其重组表达工程菌,为进一步利用其酶学活性进行抗耐药性金葡菌抑制剂的筛选奠定基础.     

志贺菌福氏2a301菌株torA基因改变的研究

目的探讨志贺菌福氏2a301菌株torA基因的改变以及与TorA(trimethylamine N-oxide TMAO reduetase，三甲胺N-氧化物还原酶)转运相关的TAT分泌系统的存在。方法利用酶活性检测和Western blot方法来验证志贺菌福氏2a301测序菌株torA基因发生的碱基置换，并且利用正常大肠杆菌中torA基因的克隆转化来研究TAT分泌系统的存在。结果实验表明志贺菌福氏2a301菌株不具有TorA酶活性，不表达TorA蛋白，和序列分析的结果相符；但是，将torA基因克隆子转化入301菌株后，转化子获得了TorA酶活性，表达了TorA蛋白。结论在志贺菌福氏2a301菌株中torA基因发生了改变，但具有完整的TAT分泌途径，可以转运蛋白。     

痢疾杆菌免疫小鼠在肠道相关淋巴组织回忆反应部位的观测

大量实验及流行病学资料证明痢疾菌感染人体后能诱导型特异免疫保护,但是否产生免疫记忆及其持续时间则所知甚少。为探索痢疾菌感染是否产生免疫记忆,作者以痢疾菌福氏2a经口服或腹腔免疫小鼠两次(间隔14天),3个月后再按原途径进行刺激。应用BA-ELISPOT法检测了小鼠派伊尔氏(PP)节结、肠系膜淋巴结(MLN)及脾脏(SPL)中特异IgA、IgG、IgM-抗体分泌细胞(Antibody secreting cell,ASC)的变化,并以MTT法测定了Con A 诱导的淋巴细胞增殖反     

人Cyclin E原核表达载体的构建及表达

目的：构建人Cyclin E基因原核表达载体,并在E.coli BL21中表达并纯化。方法：PCR扩增人Cyclin E基因片段,并将其克隆到原核表达载体pET32a（＋）中,构建重组质粒pET32a（＋）-Cyclin E。经限制性内切酶EcoRⅠ与XhoⅠ双酶切鉴定及序列测定后,转化E.coli BL21,经IPTG诱导表达组氨酸融合蛋白。结果：获得全长约为1200bp的人的Cyclin E基因片段。以构建的重组质粒pET32a（＋）-Cyclin E转化E.coli BL21后,经IPTG诱导,表达出相对分子质量（Mr）约60000的融合蛋白。经SDS-PAGE、Western blot分析显示,诱导表达的蛋白为Cyclin E。结论：成功地构建了表达载体pET32a（＋）-Cyclin E,并表达出重组蛋白His-Cyclin E,为进一步筛选在体内外能与Cyclin E和CDK2结合的新蛋白奠定了基础。     

共表达ETEC CFA／I、CS6福氏痢疾减毒株与单独表达CFA／I及CS6混合疫苗株的免疫原性比较

目的以减毒志贺菌为载体，研制肠毒素大肠杆菌(enterotoxigenic Escherichia coli，ETEC)多价疫苗可以有效防治细菌性腹泻。方法以共表达肠毒素大肠杆菌定居因子CFA/I和CS6的重组减毒福氏痢疾疫苗株FWL01(pZCF16)和两种混合的表达CFA/I的FWL01(pZHY21)和表达CS5的FWL01(pZLG6)减毒福氏痢疾疫苗株，分别以同等剂量口服免疫BMLB／c小鼠，比较二者肠毒素大肠杆菌定居因子的免疫效果。结果免疫BMLB／c小鼠全部产生了抗CFA/I和CS6的特异性血清抗体IgG和分泌型抗体sIgA，共表达CFA/I、CS6的减毒福氏痢疾菌FWL01(pZCF16)的免疫原性与混合的FWL01(pZHY21)和FWL01(pZLG6)相似或稍高。结论在同一菌株可以表达多种菌毛，构建。ETEC多价疫苗有效可行。     

利用抑制消减杂交技术(SSH)研究与Ty21a免疫相关的基因

目的:利用抑制消减杂交技术（SSH）筛选与Ty21a免疫相关的新基因。方法:以Ty21a免疫小鼠的肠细胞为tester,以正常小鼠的肠细胞为diver,提取mRNA,反转录构建cDNA文库,利用SSH技术筛选Ty21a免疫相关的新基因。结果:通过SSH技术和cDNA微矩阵技术,发现了7条与GenBank中已公布的表达序列标签（expressed　sequence tag,EST）片段没有明显同源性,可能为Ty21a免疫相关的新基因。其中3条正在用RACE-PCR法进行获得全长cDNA的工作。结论:SSH技术是一种高效的差异基因筛选方法,基因表达谱芯片技术是高通量进行基因表达模式研究的方法,Ty21a可诱导多种免疫相关新基因的表达。     

Shear Augmented Dispersion of a Solute in a Casson Fluid Flowing in a Conduit

The unsteady dispersion of a solute in a Casson fluid flowing in a conduit (pipe/channel) is studied using the generalized dispersion model of Gill and Sankarasubramanian. With this approach, the entire dispersion process is described appropriately in terms of a simple diffusion process with the effective diffusion coefficient as a function of time, in addition to its dependence on the yield stress of the fluid. The results are accurate up to a first approximation for small times, but verified with Sharp to be exact for large times. The model brings out mainly the effect of yield stress, or equivalently, the plug flow region on the overall dispersion process. It is found that the rate of dispersion is reduced (i.e., the effective diffusivity decreases) due to the yield stress of the fluid, or equivalently, the plug flow region in the conduit. Also, the effective diffusivity increases with time, but eventually attains its steady state value below a critical time [0.48(a²/D_m) for dispersion in a pipe and 0.55(a²/D_m) for dispersion in a channel—the critical transient time for a Newtonian fluid—where a is the radius of the pipe and D_m is the molecular diffusivity]. At steady state, for dispersion in a pipe with the plug flow radius one tenth of the radius of the pipe, the effective diffusivity is reduced to about 0.78 times of the corresponding value for a Newtonian fluid at equivalent flow rates; for dispersion in a channel, the reduction factor is about 0.73 confirming the earlier result of Sharp. Further, the location of the center of mass of a passive species over a cross section is found to remain unperturbed during the course of dispersion and for different values of the plug flow parameter (i.e., the yield stress of the fluid). The study can be used as a starting first approximate solution for studying the dispersion in the cardiovascular system or blood oxygenators. © 2000 Biomedical Engineering Society. PAC00: 8719Uv, 4760+i, 8350Nj, 8370Hq, 8715Vv, 8710+e, 4755Kf     

Ciliated metaplasia in a gastric adenoma in a Swedish patient

The second European case of ciliated gastric metaplasia is presented, this time in a Swedish patient. Cilia were found in cystically dilated glands underneath a gastric adenoma. The possibility that cilia represent adaptative metaplasia aimed at expelling the retained mucus within the cysts is discussed.     

Modelling a parasystolic rhythm in a heart-transplant patient

A parasystole from a heart-transplant patient is analysed using a beat-to-beat RR interval time series obtained from an electrocardiogram (ECG). The dysrhythmia, resulting from the co-existence of two pacemakers, the sinus node and an ectopic focus, presents distinctive regular patterns, with transitions from one pattern to another occurring abruptly. It is shown that the parasystolic rhythm can be simulated by a model involving two oscillators firing at fixed rates, under the restriction that neither is allowed to fire during the other's refractory period. It is found that the structure of the generated RR time series is essentially determined by the ratio of the periods of the two oscillators. In the case of a heart-transplant patient with a small heart-rate variability as a result of heart denervation, the model predicts the RR intervals with an error of less than 6% for an 80-beat sequence. From a physiological point of view, the results imply that the interaction between the two pacemakers in the heart is fairly weak, and hence the parasystole observed in the heart-transplant patient can be modelled as pure parasystole.     

Modelling a parasystolic rhythm in a heart-transplant patient

A parasystole from a heart-transplant patient is analysed using a beat-to-beat RR interval time series obtained from an electrocardiogram (ECG). The dysrhythmia, resulting from the co-existence of two pacemakers, the sinus node and an ectopic focus, presents distinctive regular patterns, with transitions from one pattern to another occurring abruptly. It is shown that the parasystolic rhythm can be simulated by a model involving two oscillators firing at fixed rates, under the restriction that neither is allowed to fire during the other's refractory period. It is found that the structure of the generated RR time series is essentially determined by the ratio of the periods of the two oscillators. In the case of a heart-transplant patient with a small heart-rate variability as a result of heart denervation, the model predicts the RR intervals with an error of less than 6% for an 80-beat sequence. From a physiological point of view, the results imply that the interaction between the two pacemakers in the heart is fairly weak, and hence the parasystole observed in the heart-transplant patient can be modelled as pure parasystole.     

Once in a lifetime: a case of a pineal tumour

This paper describes the case of a 14-year-old boy who presented to his general practitioner with a history of headaches and visual problems. Investigations subsequently revealed that he had a pineal tumour. The management of the patient is described and this rare condition discussed.     

Identification of a novel astrovirus in a domestic pig in Hungary

The family Astroviridae consists of two genera, Avastrovirus and Mamastrovirus, whose members are associated with gastroenteritis in avian and mammalian hosts, respectively. We serendipitously identified a novel porcine astrovirus in a fecal specimen from a domestic pig (Sus scrofa domestica) in Hungary. Sequencing of a fragment indicated that it was an ORF1b/ORF2/3′UTR sequence, and it has been submitted to the database as porcine astrovirus type 2 (PAstV-2/Hungary/2007) with accession number GU562296. Its unique sequence characteristics and its phylogenetic position suggest that PAstV-2 could be an important link between previously reported astroviruses and that a genetically divergent lineage of astroviruses exist in piglets.