屋尘螨抗原Der p2重组耻垢分枝杆菌的构建及其鉴定

目的构建并鉴定能够分泌表达外源蛋白——屋尘螨抗原（Der p2）的基因重组耻垢分枝杆菌（rM.s）。方法采用电穿技术,将鉴定好的PCW-Der p2质粒转化到耻垢分枝杆菌（M.s）,通过PCR鉴定目标基因在rM.s中的表达。结果经PCR鉴定,成功将PCW-Der p2质粒转化到M.s。结论成功构建了以分泌方式表达外源蛋白的Der p2-rM.s。     

结核分枝杆菌Rv0309抗原的重组表达、纯化和鉴定

目的重组表达结核分枝杆菌Rv0309蛋白,以进一步探讨其免疫效应。方法将结核分枝杆菌抗原Rv0309的全长cDNA插入到原核表达载体pGEX-4T-1中,构建成Rv0309重组质粒。将重组质粒转入大肠杆菌BL21并以IPTG诱导表达,进一步经GST亲合层析柱纯化,经SDS-PAGE和Western-blot鉴定重组表达蛋白。结果获得了pGEX4T-Rv0309重组子,并在大肠埃希菌BL21中诱导表达重组Rv0309蛋白,其条带大小约23000,与预期结果相符。结论成功地进行了结核分枝杆菌抗原Rv0309的基因克隆与重组表达,为进一步研制新型结核病疫苗打下了基础。     

铜绿假单胞菌外膜蛋白OprH原核表达载体的构建与表达

目的克隆铜绿假单胞菌外膜蛋白OprH基因,构建其原核表达载体并鉴定其表达。方法从铜绿假单胞菌中提取基因组DNA进行PCR扩增OprH基因;采用T-A克隆技术构建pMD19T-OprH重组质粒,并经酶切和序列测定后,获得OprH片段插入到原核表达载体pET28b中,以构建pET28b-OprH重组表达质粒,并在表达宿主菌E.Coli BL21中经IPTG诱导表达及通过SDS-PAGE电泳鉴定。结果 pET28b-OprH原核表达载体构建成功:重组表达质粒经IPTG诱导后表达外膜蛋白OprH。结论成功克隆了OprH基因并获得原核表达物,为进一步研究快速检测该菌的方法奠定了基础。     

牛结核分枝杆菌ESAT-6蛋白的原核表达和纯化

构建牛结核分枝杆菌ESAT-6基因的原核表达载体,诱导表达、纯化并初步鉴定该蛋白。方法:以牛型分枝杆菌基因组DNA为模板,PCR方法扩增ESAT-6基因,产物通过双酶切克隆入pET-22b(+)质粒,经PCR、酶切、测序等方法鉴定后,转入大肠杆菌BL21(DE3)polys菌体,经IPTG诱导表达蛋白;利用Western-blot鉴定重组蛋白后,纯化目的蛋白。结果:所获得ESAT-6基因序列与GenBank公布的完全一致。经SDS-PAGE分析和Western-blot鉴定均发现在6kDa处有特异性蛋白条带。纯化的蛋白的大小与蛋白质分子量标准比较一致。结论:成功表达纯化并鉴定构建了ESAT-6融合蛋白。     

大肠杆菌细菌菌蜕的制备及初步研究

目的:利用温度控制噬菌体PhiX174裂解基因E的表达,制备大肠杆菌细菌菌蜕,鉴定其裂解效率并观察其形态.方法:利用PCR技术扩增分离了噬菌体PhiX174裂解基因E并构建其表达质粒,并将质粒转化大肠杆菌DH5α构建工程菌.工程菌培养温度从28℃突升至42℃,每15 min检测菌液D600值,诱导后4 h收集菌体.体外菌落计数计算裂解效率,并通过电镜观察菌蜕结构.结果:获得273 bp的PhiX174裂解基因E全长基因及表达质粒.菌液D600在温度突升至42℃后30 min开始持续下降,2 h后基本维持不变.4 h后收集菌体测得裂解效率为99.99%.电镜观察菌蜕为具有完整外膜结构的细菌空壳,并且无细胞毒性.结论:本研究成功利用噬菌体PhiX174裂解基因E实现了大肠杆菌细菌菌蜕的制备,为进一步的疫苗及递送系统研究奠定了基础.     

表达胞壁结合型贝氏柯克斯体27 000外膜蛋白的重组卡介苗构建

目的：构建能够表达胞壁结合型贝氏柯克斯体27000外膜蛋白的重组卡介苗。方法：用PCR技术从卡介苗基因组扩增分枝杆菌19000抗原的细胞壁结合区基因和从贝氏柯克斯体基因组扩增27000外膜蛋白基因，将它们分别插入大肠杆菌-分枝杆菌穿梭质粒(pSMT3)的XbαⅠ／BamHⅠ和BarnHⅠ／HindⅢ位点。将重组的穿梭质粒转化大肠杆菌并从大肠杆菌提取重组穿梭质粒转化卡介苗。结果：从含潮霉素的培养基筛选到具有该抗生素抗性的重组卡介苗菌落，用免疫印迹分析重组质粒转化的卡介苗，发现一约27000的蛋白带与贝氏柯克斯体27000重组蛋白免疫兔血清发生特异性反应；27000重组蛋白免疫兔血清做间接免疫荧光检测重组卡介苗，结果为阳性。结论：重组卡介苗能表达贝氏柯克斯体27000外膜蛋白，表达的27000外膜蛋白存在于卡介苗菌体表面，卡介苗菌体表面的27000抗原可能诱导抗Q热免疫应答。     

结核分枝杆菌Rv2389c基因的克隆和原核表达

目的:克隆并原核表达结核分枝杆菌Rv2389c基因。方法:培养结核分枝杆菌,提取其基因组,PCR扩增出485bp的Rv2389c基因,克隆入pSP73载体,测序分析。将该目的基因亚克隆入pGEX-4T-2表达载体,测序证实正确后转化大肠杆菌BL21(DE3),待大肠杆菌增菌至A600nm值为0.6时,用0.3 mmol/L IPTG在30℃诱导表达重组蛋白。结果:测序结果证实获得了Rv2389c基因,其序列与GenBank中报道完全一致。构建了重组表达质粒,表达的蛋白以12%SDS-PAGE分析,在Mr约42KD处出现一条新生蛋白带,经凝胶薄层扫描检测,表达量约占菌体蛋白的26.8%。结论:成功克隆了结核分枝杆菌Rv2389c基因并得到了其大肠杆菌表达产物,为进一步研究其活性和功能奠定了基础。     

重组腺病毒Ad-huVEGF121的制备

目的：构建能够用于骨缺血研究的能表达huVEGF121的重组腺病毒载体。方法：将huVEGF121克隆至穿梭质粒，线性化后转染含骨架质粒的BJ5183菌，构建重组腺病毒质粒。酶切及PCR法鉴定；将其线性化后转染293细胞，通过观察绿色荧光监测病毒产生，免疫组化检测VEGF121表达。结果：成功的将hu—VEGF121克隆至穿梭质粒pTrack—CMV，正确构建了重组腺病毒质粒，感染293细胞得到大量病毒。结论：成功构建了huVEGF121重组腺病毒，为缺血性骨病的进一步研究奠定了基础。     

尼帕病毒N基因的原核表达

目的利用尼帕病毒(NiV)的N基因进行原核表达,探讨NiV N蛋白作为检测NiV存在的抗原的可能性。方法利用pET-28a(+)作为表达载体,构建pET-NiV N重组质粒,通过IPTG诱导使其表达蛋白,并用Western blot方法进行原核表达NiV N蛋白的检测。结果通过PCR、酶切和连接反应,成功构建N蛋白的pET-NiV N重组质粒,大小为1596 bp;利用IPTG诱导pET-NiV N重组质粒在宿主菌中表达N蛋白,SDS-PAGE结果显示,55 k D有特异蛋白条带,与蛋白预计大小相符,经Bandscan软件扫描分析,重组蛋白量占菌体总蛋白量的12%;Western blot法检测显示,55 k D的条带显色良好,证明是本实验所需要得到的目的蛋白。结论使用原核表达系统成功表达了NiV的N蛋白,为其进一步的抗体制备、活性鉴定及研究应用奠定了基础。     

菌落PCR快速分析抗体库重组率时的假阳性现象

目的　评价菌落PCR法鉴定噬菌体抗体库阳性重组率的可靠性。方法　鼠抗体基因Lc片段、Fd片段经酶切、纯化后 ,依次克隆入pComb3载体上相应的酶切位点 ,电转化XL1 blue菌后建立鼠源性Fab噬菌体抗体库。比较菌落PCR法、质粒PCR法及质粒酶切法鉴定转化菌阳性重组率的一致性。同时用空菌、空载体、空载体菌等作模板为对照PCR ,以排除相关组分的干扰。结果　建立的Lc库的库容为 1.175×10 6CFU ,Fab库的库容为 1.0 2×10 6CFU。 3种方法鉴定的阳性重组率不同 (Lc的重组率依次为 10 0 %、78%、78% ,Fd的重组率依次为 90 %、6 6 %、6 6 % ,Lc与Fd同时插入的重组率依次为 90 %、5 0 %、5 0 % )。菌落PCR法鉴定的重组率偏高 ,存在假阳性现象。对照PCR提示 ,XL1 blue菌本身可能是导致假阳性扩增的原因。结论　用菌落PCR法不能准确鉴定噬菌体抗体库的重组率 ,用质粒PCR法或质粒酶切法鉴定更为可靠     

上尿路结石磁共振仿真输尿管肾镜检查的实验研究

目的 :探讨源于 4种磁共振序列的仿真输尿管肾镜成像诊断上尿路结石的价值。材料和方法 :将 9枚直径 1～5mm的尿路结石经输尿管断端置入离体输尿管肾盂内 ,分别以生理盐水、3 %马根微显溶液充盈该上尿路后 ,行HT2 FSE、多层薄层SS FSE和 3D、2DFSPGR序列扫描。源影像在工作站用Navigator软件作内镜成像 (VE)重建。结果 :VE对结石的敏感性源于多层薄层SS FSE序列者为 4/ 9、源于HT2 FSE序列者 5 / 9、源于 3D和 2DFSPGR序列者均为 4/ 9,检出结石最小直径为 2mm。VE所示结石形态、大小与实际略有出入。源于HT2 FSE序列的VE图像质量最佳 ,其次依次多层薄层SS FSE序列、3D和 2DFSPGR序列 (P <0 .0 5 )。结论 :磁共振仿真输尿管肾镜能够检出直径 2mm以上尿路结石 ,以HT2 FSE序列采集源影像为佳。     

The value of MR angiography techniques in the detection of head and neck paragangliomas

OBJECTIVE: The objective of this study was to compare three-dimensional phase-contrast angiography (3D PCA), 2D time-of-flight (2D TOF), and 3D TOF magnetic resonance (MR) angiography and a proton density weighted technique in terms of their ability to detect head and neck paragangliomas. MATERIALS AND METHODS: 14 patients with 29 paragangliomas were examined at 1.5 T. Three MR angiography sequences (3D PCA, 2D TOF, and multi-slab 3D TOF) and a proton density (PD) weighted sequence were reviewed by four neuroradiologists. The gold standard was digital subtraction angiography. Presence of tumor was assessed in five grades of confidence. Sensitivity and specificity were calculated after dichotomizing the results. Data was analyzed using the logistic regression method. RESULTS: Mean sensitivity and specificity for the four observers were for PD: 72%/97%, for 3D PCA: 75%/90%, for 2D TOF: 66%/93%, and for 3D TOF: 90%/92%. Sensitivity was significantly better for 3D TOF MRA (P < 0.001). No substantial between-observer variation for tumor detection was present. CONCLUSION: Our results demonstrate that, using 3D TOF MRA, paragangliomas in the head and neck region can be detected with high sensitivity and specificity. Further investigation is necessary to judge the value of 3D TOF MR angiography against fat suppressed contrast enhanced T1 weighted and fat suppressed T2 weighted MR sequences to find the optimal imaging sequence for paragangliomas.     

Articular cartilage defects detected with 3D water-excitation true FISP: prospective comparison with sequences commonly used for knee imaging

PURPOSE: To prospectively compare the accuracy of three-dimensional (3D) water-excitation (WE) true fast imaging with steady-state precession (FISP) in the diagnosis of articular cartilage defects with that of sequences commonly used to image the knee, with arthroscopy or surgery as the reference standard. MATERIALS AND METHODS: This study protocol was institutional review board approved. Written informed consent was obtained from all patients. Thirty knees in 29 patients (mean age, 56 years; range, 18-86 years) were prospectively evaluated by using sagittal 3D WE true FISP with two section thicknesses (1.7 mm [true FISPthin] and 3.0 mm [true FISPthick]), two-dimensional (2D) intermediate-weighted spin-echo with fat saturation, 2D fast short inversion time inversion-recovery, 3D WE double-echo steady-state, and 3D fat-saturated fast low-angle shot sequences. Cartilage defects were graded on magnetic resonance images and during surgery with a modified Noyes scoring system. Contrast-to-noise ratio (CNR) and CNR efficiency were calculated. Sensitivity, specificity, and accuracy were assessed. Interobserver agreement was determined with kappa statistics, and quantitative results were evaluated with the Wilcoxon signed rank test. RESULTS: The performance of 3D WE true FISPthick (sensitivity, specificity, and accuracy, respectively, were 52%, 93%, and 71% for reader 1 and 65%, 88%, and 76% for reader 2) and 3D WE true FISPthin (sensitivity, specificity, and accuracy, respectively, were 58%, 94%, and 75% for reader 1 and 63%, 80%, and 71% for reader 2) sequences was no different than that of other sequences in the detection of circumscribed defects. Three-dimensional WE true FISP sequences had a significantly (P<.0033) higher CNR and CNR efficiency between cartilage and fluid than the corresponding sequences with the same section thickness. CONCLUSION: Three-dimensional WE true FISP enables high contrast between joint fluid and articular cartilage and a diagnostic performance that is comparable with that of standard sequences.     

Visualization of cranial nerves I–XII: value of 3D CISS and T2-weighted FSE sequences

The aim of this study was to evaluate the sensitivity of the three-dimensional constructive interference of steady state (3D CISS) sequence (slice thickness 0.7 mm) and that of the T2-weighted fast spin echo (T2-weighted FSE) sequence (slice thickness 3 mm) for the visualization of all cranial nerves in their cisternal course. Twenty healthy volunteers were examined using the T2-weighted FSE and the 3D CISS sequences. Three observers evaluated independently the cranial nerves NI–NXII in their cisternal course. The rates for successful visualization of each nerve for 3D CISS (and for T2-weighted FSE in parentheses) were as follows: NI, NII, NV, NVII, NVIII 40 of 40 (40 of 40), NIII 40 of 40 (18 of 40), NIV 19 of 40 (3 of 40), NVI 39 of 40 (5 of 40), NIX, X, XI 40 of 40 (29 of 40), and NXII 40 of 40 (4 of 40). Most of the cranial nerves can be reliably assessed when using the 3D CISS and the T2-weighted FSE sequences. Increasing the spatial resolution when using the 3D CISS sequence increases the reliability of the identification of the cranial nerves NIII–NXII. Received: 29 September 1999; Revised: 2 February 2000; Accepted: 21 March 2000     

RNA干扰沉默cyclin D1基因表达对乳腺癌细胞迁移和侵袭力的影响

 目的 利用RNA干扰技术下调cyclin D1基因表达,探讨其对乳腺癌细胞体外侵袭和迁移能力的影响,了解cyclin D1在肿瘤细胞转移的作用.方法 设计cyclin D1基因的小分子干扰RNA(small interfering RNA,siRNA)片段siRNA51和siRNA52,脂质体介导转染入乳腺癌MDA-MB-231细胞株.应用Western印迹法检测转染前后cyclin D1表达水平;通过单层细胞划痕实验观察细胞转染前后迁移能力的变化,Transwell侵袭实验检测细胞体外侵袭力.结果 乳腺癌MDA-MB-231细胞株转染cyclin D1的siRNA后,cyclin D1蛋白水平明显下调,细胞的迁移及侵袭能力明显减弱.结论 利用RNA干扰技术能够特异阻断cyclin D1基因表达;cyclin D1基因表达下调能够明显抑制乳腺癌细胞的迁移及侵袭能力.     

脑血管畸形磁共振血管成像与脑血管造影对照研究

目的：探讨了ＴＯＦ法ＭＲＡ评价脑血管畸形的价值及限度，材料与方法：２９例经ＭＲＩ（ｎ＝２９），脑血管造影（ｎ＝１５）和／或手术病理（ｎ＝１０）证实的脑血管畸形（ＡＶＭ２４例，ＡＶＦ２例，隐匿性ＡＶＭ３例）患者进行ＴＯＦ法ＭＲＡ检查，应用１．０Ｔ超导ＭＲ系统的二维或三维ＦＩＳＰ序列（预置０～３条预饱和带）和ＭＩＰ图像后处理技术，１５例同时经股动脉行全脑血管造影，采用Ｋａｐｐａ统计量对两种血管成像方法     

Evaluating contrast kinetics by acquiring 2D images during 3D contrast-enhanced MR angiography

PURPOSE: To monitor contrast kinetics by acquiring multiple 2D images during 3D contrast-enhanced magnetic resonance angiography (CE MRA). MATERIALS AND METHODS: A 2D real-time autotriggering tool was integrated into a 3D sequence, enabling it to run multiple times during 3D acquisition. Several dummy scans were applied after each transition to maintain the steady state condition of both sequences. The number of the acquired 2D images and their distribution can be adjusted. Each 2D image was saved along with its associated timing. Contrast signal variations over time were plotted, reflecting selective signal measurement over an artery and vein from the saved 2D images. RESULTS: Different contrast kinetics timings were calculated from the resulting plot. Contrast arrival time to the internal cerebral artery was 13.2 +/- 1.2 seconds and the peak arterial to peak venous (at the confluence of sinuses) enhancement was 6.7 +/- 0.6 seconds. The observed timing could be used for 3D sequence optimization; the saved 2D images are useful in detecting and characterizing vascular abnormalities. CONCLUSION: Integrating 2D and 3D sequences into one sequence to monitor contrast kinetics through the neurovasculature is feasible without the need for extra injections or reduced spatial resolution. The technique can also be used in different parts of the body to extract useful clinical information.     

Contrast-enhanced MR angiography in patients with carotid artery stenosis: comparison of two different techniques with an unenhanced 2D time-of-flight sequence

Conventional time-of-flight (TOF) MR angiography (MRA) in carotid artery stenosis relies on flow-related enhancement to produce signal from vascular structures. Intravoxel phase dispersion, due to vortices, causes loss of signal and is the reason for the tendency to overestimate the degree of stenosis. In contrast-enhanced MRA, intravascular signal is mainly dependent on T1 shortening of the blood. We compared first-pass contrast-enhanced MRA (contrast-enhanced 3D gradient echo, ce3D GRE) and contrast-enhanced 2D TOF (ce2D TOF) sequences with an unenhanced 2D TOF in 13 patients with carotid artery stenosis, assessing delineation of the carotid bifurcation, enhancement of veins and grade of stenosis. The contrast-enhanced techniques produced more morphological detail, the ce3D GRE being superior to the ce2D TOF. Four carotid arteries were reclassified into lesser stenosis categories using the ce3D GRE technique. However, seven carotid arteries (27 %) were rated as nondiagnostic on the ce3D GRE, mainly due to masking of the carotid bifurcation by veins. The latter can be avoided by decreasing the acquisition time; on our 1.5-T system we could achieve a minimum time of 23 s per 3D GRE. Further reduction of acquisition time would be necessary to incorporate this method into clinical routine, requiring higher-performance gradients, which are not available in many UK hospitals. Received: 25 February 1999 Accepted: 29 July 1999     

A 3D T1-weighted gradient-echo sequence for routine use in 3D radiosurgical treatment planning of brain metastases: first clinical results

The authors report on a 3D sequence for MRI of the brain and its application in radiosurgical treatment planning of 35 brain metastases. The measuring sequence, called magnetization — prepared rapid gradient echo (MPRAGE), was compared with 2D T1-weighted spin-echo (SE) sequences following intravenous contrast-medium application in 19 patients with brain metastases. The average diameter of all lesions was similar in both sequences, with 16.8 and 17.0 mm for SE and MPRAGE, respectively. Target point definition was equal in 29 metastases, and in 6 cases superior on MPRAGE, due to better gray-white matter contrast and increased contrast enhancement. In cases of bleeding metastases there was improved depiction of internal structures in 3D MRI. Postprocessing of 3D MPRAGE data created multi-planar reconstruction along any chosen plane with isotropic spatial resolution, which helped to improve radiosurgical isodose distribution in 4 cases when compared to 2D SE. However, sensitivity of 3D MPRAGE to detect small lesions (< 3 mm) was decreased in one patient with more than 50 metastases. We conclude that 3D gradient-echo (GE) imaging might be of great value for radiosurgical treatment planning, but does not replace 2D SE with its current parameters. Correspondence to: H. Hawighorst