结核分枝杆菌突变gyrase A基因原核表达载体的构建与鉴定

目的构建结核分枝杆菌Ser95Thr突变DNA促旋酶A(DNA gyrase A)基因原核表达载体并鉴定,为进一步研究奠定基础。方法以结核分枝杆菌H37Rv基因组为模板,应用重叠延伸剪切技术,分别通过3次PCR扩增Ser95Thr突变gyrase A基因编码序列,定向克隆入融合蛋白原核表达载体pET-32 a(+),获得重组表达质粒pET-mgyr。结果从结核分枝杆菌H37Rv株基因组DNA中扩增出Ser95Thr突变gyrase A基因,经过酶切、PCR和测序鉴定,表明突变gyrase A基因正确地插入原核表达载体pET-32 a(+)。结论成功构建了原核表达载体pET-mgyr,为Ser95Thr突变gyrase A基因的功能研究奠定了基础。     

含KSHV vIL-6基因原核表达载体的构建及融合蛋白的表达纯化与鉴定

目的:在大肠埃希菌中表达卡波济肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus,KSHV)vIL-6基因,并进一步纯化和鉴定表达蛋白.方法:以本实验室已构建的重组质粒pGEX-6p-1-vIL-6为模板,PCR扩增目的基因片段,将PCR产物克隆至原核表达载体pET-32a(+)中,构建vIL-6的重组原核表达质粒pET-32a(+)-vIL-6.将重组质粒转化大肠埃希菌(E.coli)BL21(DE3)感受态细胞,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白.表达产物通过镍亲和层析柱纯化并经蛋白质印迹法鉴定.结果:限制性内切酶检测和基因测序证实,成功构建了含有vIL-6基因的原核表达载体.蛋白质印迹法结果显示,在大肠埃希菌BL21(DE3)中His-Tag融合蛋白得到了表达,亲和层析法纯化后获得了相对分子质量为43 000的vIL-6融合蛋白.结论:重组质粒pET-32a(+) -vIL-6能在大肠埃希菌BL21(DE3)中表达,利用镍亲和层析柱可纯化获得融合蛋白.     

人组织型谷氨酰胺转氨酶基因的克隆 表达及纯化

目的:克隆人组织型谷氨酰胺转氨酶基因,并进行原核表达及纯化.方法:从Caco-2细胞中提取总RNA,利用RT-PCR克隆人组织型谷氨酰胺转氨酶基因,然后将其连接到原核表达载体pET-32a.将构建成功的重组表达载体pET-32a-TG2导入大肠杆菌BL21(DE3),IPTG诱导表达His-TG2融合蛋白.用Ni-NTA蛋白纯化柱对融合蛋白进行纯化,采用Western Blotting检测重组蛋白的特异性.结果:成功克隆出人组织型谷氨酰胺转氨酶基因,构建了pET-32a-TG2原核表达载体,并成功诱导了重组融合蛋白His-TG2的表达.结论:构建了表达人组织型谷氨酰胺转氨酶基因的原核表达系统,并成功对重组蛋白进行了纯化.     

小鼠Foxp3蛋白的原核表达及多克隆抗体的制备

目的:体外原核表达小鼠Foxp3蛋白,并以其免疫家兔,制备多克隆抗体。方法:从质粒Foxp3-pMD-18T扩增小鼠Foxp3全长基因,亚克隆至原核表达载体pET-32 a,转化E.coliBL-21;IPTG诱导表达目的蛋白,N i2 -NTA柱纯化及蛋白质印迹鉴定蛋白;纯化蛋白免疫家兔,制备多克隆抗体,ELISA法检测抗体效价,蛋白质印迹检测抗体特异性。结果:成功构建了Foxp3-pET-32 a原核表达载体,表达和纯化了目的蛋白;ELISA法检测抗体效价为1∶20 000,蛋白质印迹检测结果显示抗体特异性与小鼠Foxp3蛋白结合。结论:成功表达小鼠Foxp3蛋白并制备了多克隆抗体。     

登革2型病毒NGC株E基因部分序列原核蛋白表达

目的:构建登革2型病毒NGC株E基因区1～476 bp的原核表达载体并进行原核表达.方法:将登革2型病毒NGC株E基因区部分序列克隆入原核表达载体pET28a(+),命名为pET28a(+)-En;经酶切、PCR及测序鉴定后转化BL21(DE3)菌株,用IPTG诱导表达,通过SDS-PAGE、Western印迹鉴定表达蛋白;对表达蛋白进行纯化,并滴定对C6/36细胞的TCID50.结果:(1)成功构建了pET28a(+)-En原核表达重组质粒,SDS-PAGE分析表明,E基因区部分序列获得高效表达,相对分子量约为23 kD,表达量约占菌体总蛋白的29%;Western印迹表明该目的蛋白可与登革2型病毒鼠单克隆抗体结合;(2)用Ni柱亲和层析法纯化原核表达蛋白,纯度达90%;(3)DEN-2 NGC株E基因部分序列原核表达蛋白对C6/36细胞的TCID50为10-4.88/0.1 ml.结论:pET28a(+)-En可在BL21(DE3)菌株中高效表达,DEN-2 NGC株E基因部分序列原核表达蛋白对C6/36细胞有一定的细胞毒作用.     

融合蛋白表达载体的构建及在大肠杆菌中的表达

目的构建人类regucalc in重组原核表达载体。方法用RT-PCR的方法从人类肝癌细胞系HepG2中扩增出regucalc in基因cDNA序列,应用T克隆载体及pET-32 a(+)表达载体后,将质粒转化进入原核表达菌株进行表达。结果表达产物经SDS-PAGE电泳分析后,在45 000处有一明显表达条带,与理论结果相符。结论成功构建融合表达载体Pet-regucalc in,并在原核细胞中表达出人类regucalc in-H is融合蛋白。     

家蚕蚕蛹过敏原几丁质酶基因的克隆和原核表达载体的构建

目的 克隆、表达家蚕蚕蛹过敏原几丁质酶基因,并构建其原核表达载体.方法 提取家蚕的总RNA,RT-PCR克隆家蚕过敏原几丁质酶的全长基因,设计简并引物,扩增家蚕几丁质酶基因的完整开放阅读框,与pET-28a载体连接,构建原核表达载体.结果 成功克隆家蚕主要变应原几丁质酶基因并构建其原核表达载体.该基因含有长度为1 668 bp的开放阅读框,编码555个氨基酸.该蛋白质的相对分子质量为61 500,电点为5.78,编码序列与数据库中已知的家蚕几丁质酶基因的同源性为99%.结论 成功克隆家蚕蚕蛹主要过敏原几丁质酶基因并构建了原核表达载体,为家蚕蚕蛹过敏原几丁质酶的重组表达和免疫活性鉴定等奠定了基础.     

点带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达

目的克隆点带石斑鱼hepcidin前体cDNA序列,构建其成熟肽pET-32a融合表达载体。结论根据已报道的硬骨鱼类hepcidin cDNA序列设计简并引物,以点带石斑鱼(Epinephelelus malabaricus)肝脏为材料,通过RT-PCR扩增hepcidin cDNA序列,用比对推导的氨基酸序列预测其成熟肽段。再将成熟肽cDNA序列亚克隆至pET-32a构建原核表达并进行原核融合表达。结果获得点带石斑鱼hepcidin-like抗菌肽前体cDNA序列1条,GenBank登录号为HM474788。DNA序列的Blast分析以及推导氨基酸序列NJ进化树分析表明,HM474788是点带石斑鱼第1条报道的抗菌肽hepcidin cDNA序列。成功构建了该序列的成熟肽原核融合表达载体pET-32a-hepcidin(EM1),并成功在大肠杆菌origami(DE3)中表达。结论成功克隆点带石斑鱼hepcidin-like抗菌肽前体cDNA序列,成熟肽成功在大肠杆菌中融合表达,为后续有关石斑鱼hepcidin的功能研究与实践应用奠定重要基础。     

羊布鲁氏菌omp25基因原核表达载体的构建与鉴定

目的:利用PCR反应从羊布鲁氏菌基因组DNA中克隆omp25基因,并将其构建到原核表达载体pET-32a中。方法:试剂盒法提取羊布鲁氏菌基因组DNA,设计合适的引物,通过PCR反应从基因组中扩增外膜蛋白OMP25的编码基因(omp25)。将得到的基因片段连接到克隆载体pMD19-T的多克隆位点(MCS)中,将连接体pMD19-T-omp25转化感受态大肠杆菌DH5(并进行筛选和克隆扩增,提取质粒并进行酶切分析与序列测定。利用酶切与连接反应将目的基因(omp25)插入载体pET-32a的多克隆位点中,将连接体pET-32a-omp25转化感受态大肠杆菌DH5(并进行筛选和克隆扩增,提取质粒并进行酶切分析。结果:PCR扩增得到的基因片段与GenBank中已公布的羊布鲁氏菌omp25基因同源性为99%。酶切分析结果表明,omp25基因成功地插入载体pET-32a中。结论:成功地扩增得到了羊布鲁氏菌omp25基因并构建了携带该基因的重组原核表达载体pET-32a-omp25,为后续的研究提供了可靠的基础。     

人瘦素基因的原核表达载体构建及其蛋白结构分析

目的 克隆人源Leptin的成熟肽编码序列后基因并构建其原核重组表达菌株,对其产物进行纯化鉴定;生物信息学分析其蛋白质结构并预测功能.方法 提取人脂肪干细胞中的总RNA,经RT-PCR反转录获得cDNA序列,PCR扩增Leptin基因并将其克隆入表达载体pET-28a(+)原核质粒,构建该基因的重组原核表达载体pET-28a-Leptin.随后进行双酶切和测序鉴定.在E.coli BL21(DE3)中诱导表达目的蛋白,通过WB检测Leptin蛋白的表达;利用在线网络生物信息学相关数据库和分析软件对测序结果进行分析,并对重组蛋白结构及功能等进行验证和预测.结果 PCR扩增得到Leptin目标DNA片段;成功构建重组表达质粒pET-28a-Leptin,经IPTG诱导表达后,目的蛋白在宿主E.coli BL21 (DE3)中高效表达Leptin融合蛋白,相对分子质量18000.生物信息学检索该基因编码蛋白的氨基酸序列与理化参数显示,蛋白无跨膜区,有6个Ser、1个Thr可能为蛋白激酶磷酸化位点,第1～21位可能为信号肽区域.在Leptin氨基酸序列中:α-螺旋93处,无规则卷曲43处,延伸链17处,β-转角14处,分别占总二级结构的55.69％、25.75％、10.18％和8.38％.结论 成功克隆了人的Leptin基因.利用原核载体表达系统成功表达并进一步纯化的Leptin蛋白具有免疫原性.其生物信息学分析及结合蛋白质结构与功能预测结果可为深入研究Leptin蛋白的活性作用提供有益借鉴.     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.