结核分枝杆菌ESAT-6基因的克隆与表达

目的 对结核分枝杆菌的ESAT-6基因进行克隆、鉴定与表达，为结核病的诊断、重组疫苗的应用打下基础。方法 以结核分枝杆菌H37Rv株基因组DNA为模板，用聚合酶链反应(PCR)对ESAT-6基因进行扩增，将扩增的产物连接于测序载体pGEM-T上，经测序反应确定无误后，再将PCR产物与原核表达载体pET42b( )构建表达ESAT-6的重组质粒，将重组质粒先转化人大肠杆菌DH5a内并提取质粒，经酶切鉴定后，再转化入表达宿主大肠杆菌BL21菌株内，对转化菌株进行诱导后，破菌，进行SDS-PAGE电泳。结果 电泳发现转化了重组质粒的菌株有表达蛋白，其表达的蛋白质相对分子质量为9900。结论 目的基因克隆到菌株内，重组结核分枝杆菌ESAT-6的成功表达为结核病诊断、重组疫苗应用和免疫效应检测等奠定了基础。     

副溶血弧菌TDH基因的克隆与表达

目的对副溶血弧菌TDH基因进行克隆、鉴定与表达，为制备单抗、诊断试剂及进一步深入研究TDH的功能奠定基础。方法用聚合酶链反应(PCR)扩增TDH基因将扩增的产物连接于测序载体pGEM-T上，经测序反应确定无误后，再将PCR产物与原核表达载体pQE100构建表达TDH的重组质粒，将重组质粒先转化人大肠杆菌DH5a内并提取质粒，经双酶切鉴定后，再转化入表达宿主大肠杆菌BL21菌株内，对转化菌株进行诱导后，破菌，进行SDS-PAGE电泳。结果电泳发现转化了重组质粒的菌株有表达蛋白，其表达的蛋白质相对分子质量为24000。结论TDH基因成功克隆到表达质粒内并表达，为制备单抗、诊断试剂与其致病机制研究奠定了基础。     

副溶血弧菌TDH基因的克隆与表达

目的　对副溶血弧菌TDH基因进行克隆、鉴定与表达 ,为制备单抗、诊断试剂及进一步深入研究TDH的功能奠定基础。方法　用聚合酶链反应 (PCR)扩增TDH基因将扩增的产物连接于测序载体 pGEM T上 ,经测序反应确定无误后 ,再将PCR产物与原核表达载体 pQE10 0构建表达TDH的重组质粒 ,将重组质粒先转化入大肠杆菌DH5α内并提取质粒 ,经双酶切鉴定后 ,再转化入表达宿主大肠杆菌BL2 1菌株内 ,对转化菌株进行诱导后 ,破菌 ,进行SDS PAGE电泳。结果　电泳发现转化了重组质粒的菌株有表达蛋白 ,其表达的蛋白质相对分子质量为 2 4 0 0 0。结论　TDH基因成功克隆到表达质粒内并表达 ,为制备单抗、诊断试剂与其致病机制研究奠定了基础。     

esat6-卡介苗重组疫苗的构建及鉴定

目的:获得结核分枝杆菌esat6-卡介苗重组疫苗。方法:通过基因工程重组技术将结核分枝杆菌保护性抗原esat6的编码基因与穿梭质粒载体pYUB295重组,通过电穿孔技术导入卡介苗中,应用PCR扩增、PAGE电泳鉴定重组卡介苗。结果:通过PCR扩增获得300bp左右的esat6基因,与穿梭质粒载体pYUB295重组后,通过PCR扩增、限制性内切酶酶切、DNA测序鉴定表明成功地构建了esat6基因pYUB295重组质粒。将重组质粒通过电穿孔导入卡介苗,重组卡介苗在抗性培养基上生长良好。pYUB295-esat6-卡介苗重组疫苗基因组DNA的PCR扩增以及培养上清液的PAGE电泳表明esat6-卡介苗重组疫苗构建正确,esat6蛋白在卡介苗中分泌表达。结论:成功构建了esat6-卡介苗重组疫苗。     

结核分枝杆菌ESAT6基因克隆与表达质粒构建

目的　克隆结核分枝杆菌 (MTB)分泌蛋白ESAT6基因 ,并构建重组表达质粒。方法　根据Genbank中ESAT6基因序列 ,针对其编码区合成引物 ,采用PCR方法从结核分枝杆菌H37Rv基因组DNA中扩增出ESAT6基因 ,并连接到T载体 ,然后定向克隆到原核表达质粒pGEX 4T 2 ,阳性克隆用酶切和DNA测序鉴定。结果　经双内切酶消化所切下的片段 ,大小与预计相符 ;测序结果证实基因序列正确 ,符合表达框架。结论　成功构建了重组原核表达质粒 pGEX ESAT6。     

结核分枝杆菌ESAT6基因克隆与表达质粒构建

目的 克隆结核分枝杆菌(MTB)分泌蛋白ESAT6基因，并构建重组表达质粒。方法 根据Genbank中ESAT6基因序列，针对其编码区合成引物，采用PCR方法从结核分枝杆菌H37Rv基因组DNA中扩增出ESAT6基因，并连接到T载体，然后定向克隆到原核表达质粒pGEX-4T-2，阳性克隆用酶切和DNA测序鉴定。结果 经双内切酶消化所切下的片段，大小与预计相符；测序结果证实基因序列正确，符合表达框架。结论 成功构建了重组原核表达质粒pGEX-ESAT6。     

金黄色葡萄球菌肠毒素A基因克隆及表达

目的对金黄色葡萄球菌肠毒素A基因(SEA)进行克隆、鉴定与表达,为制备单抗、诊断试剂及进一步深入研究SEA的功能奠定基础。方法用聚合酶链反应(PCR)扩增SEA基因,将扩增的产物连接于测序载体pMD18-T上,经测序反应确定无误,酶切后将SEA基因与原核表达载体pET42b( )构建表达SEA的重组质粒,将重组质粒先转化入大肠杆菌DH5α内并提取质粒,经双酶切鉴定后,再转化入表达宿主大肠杆菌BL21菌株内,对转化菌株进行诱导后,破菌,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)。结果构建了表达载体pET42b( )-SEA,并获得高效表达,表达的蛋白质相对分子质量为27 000,重组蛋白(rSEA)在37℃、25℃经异丙基硫化-β-D半乳糖苷(IPTG)诱导时均以包涵体形式存在。结论SEA基因成功克隆到表达质粒内并表达,为制备单抗、诊断试剂及其致病机制研究奠定了基础。     

金黄色葡萄球菌肠毒素A基因克隆及表达

目的 对金黄色葡萄球菌肠毒素A基因（SEA）进行克隆、鉴定与表达，为制备单抗、诊断试剂及进一步深入研究SEA的功能奠定基础。方法 用聚合酶链反应（PCR）扩增SEA基因，将扩增的产物连接于测序载体pMD18-T上，经测序反应确定无误，酶切后将SEA基因与原核表达载体pET42b（＋）构建表达SEA的重组质粒。将重组质粒先转化入大肠杆菌DH5a内并提取质粒，经双酶切鉴定后，再转化入表达宿主大肠杆菌B121菌株内，对转化菌株进行诱导后，破菌，进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）。结果 构建了表达载体pET42b（＋）-SEA，并获得高效表达，表达的蛋白质相对分子质量为27000，重组蛋白（rSEA）在37℃、25％经异丙基硫化-β-D半乳糖苷（IPTG）诱导时均以包涵体形式存在。结论 SEA基因成功克隆到表达质粒内并表达，为制备单抗、诊断试剂及其致病机制研究奠定了基础。     

金黄色葡萄球菌N-乙酰甘露糖胺-6-磷酸2-异构酶基因的克隆与表达

目的对金黄色葡萄球菌N-乙酰甘露糖胺-6-磷酸2-异构酶基因(APE)进行克隆、鉴定与表达,为新型抗菌药物的设计提供参考依据。方法采用聚合酶链反应(PCR)扩增APE基因,将扩增产物酶切后与原核表达载体pET-32a(+)连接,构建表达APE的重组质粒,将重组质粒先转化大肠杆菌TG1内并提取质粒,经PCR、双酶切鉴定后再转化表达宿主大肠杆菌BL21(DE3),对转化菌株进行诱导后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。结果构建了表达载体pET-32a(+)-APE,在37℃经异丙基-β-D硫代半乳糖苷诱导时获得表达,表达的蛋白质相对分子质量为45.2×10~3。结论 APE基因成功克隆到表达质粒内并获得了表达。     

构建Loa22基因去信号肽片段原核重组表达载体

目的：构建赖型钩端螺旋体OmpA膜蛋白Loa22基因去信号肽片段的原核表达载体，并对其进行克隆表达。方法：实验于2004—12／2005—12在四川大学华西医学中心感染免疫研究室完成。以赖型钩端螺旋体017株基因组DNA为模板，PCR扩增Loa22基因去信号肽片段，亚克隆至原核表达载体pGEX-4T-1，经双酶切、PCR鉴定，筛选出阳性重组质粒克隆。经DNA测序正确后，转化大肠杆菌，利用IPTG进行诱导表达，通过SDS—PAGE鉴定表达产物。结果：PCR获得长516bp的片段。Loa22基因去信号肽片段与pGEX-4T-1的重组质粒构建成功。重组质粒经IPTG诱导后能在大肠杆菌中表达Mr45000的融合蛋白。结论：制备了Loa22基因去信号肽片段原核重组表达载体，为钩体新型疫苗的研究奠定基础。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

手转胎头术失败的原因与分娩结局临床分析

目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4％(55/72)]明显高于成功组[31.7％(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3％(42/72)]高于成功组[38.1％ (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9％ (28/72)]高于成功组[23.8％(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大＜6 cm、胎头位于坐骨棘上及宫口开大8～10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1％(44/72)]高于成功组[38.9％(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2％(52/72)]明显高于成功组[34.1 ％(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.