流行性乙型脑炎／登革病毒1型嵌合基因的构建和鉴定

目的：构建和鉴定流行性乙型脑炎（乙脑）／登革病毒1型（ DV1）／JEV prME嵌合基因。方法利用分子生物学技术构建登革病毒和乙脑病毒嵌合的prME嵌合基因,重叠PCR法鉴定;在质粒载体pcDNA3．1（＋）的基础上,构建表达pcDV1／JEV prME的重组质粒载体,利用LipofectamineTM2000将登革／乙脑prME嵌合质粒转染至BHK－21细胞,间接免疫荧光法检测prME蛋白在细胞中的表达。结果 DNA测序结果证实pcDV1／JEV prME重组质粒完全正确,成功构建的登革／乙脑嵌合质粒在BHK－21细胞中能够表达prME蛋白。结论通过一系列的鉴定手段,可以认定本实验中构建的登革／乙脑嵌合质粒能够在真核细胞中顺利表达。     

乙型脑炎病毒结构蛋白C＋pre M和E基因的克隆与序列分析

目的 对乙型脑炎病毒(Japanese encephalitis virus, JEV)结构蛋白基因进行扩增、克隆和序列测定.方法 根据GenBank中发表的乙脑病毒结构蛋白基因C + pre M基因和E 序列,设计合成两对引物,以JEV Nakayama-NIH株RNA为模板,反转录并扩增目的 cDNA,然后与 pGEM-T easy载体连接并转化大肠杆菌DH5α菌株,提取的重组质粒用电泳、PCR和酶切鉴定后并进行测序.将JEV Nakayama-NIH与GenBank中报道的多株JEV结构蛋白C + pre M 基因和E基因序列进行了比较.结果 Nakayama-NIH与其他参考株C + pre M和E基因的核苷酸同源性为99%,证实扩增克隆的是JEV的结构蛋白C + pre M和E基因.结论 对JEV分子生物学特征进行了分析,为JEV特异性核酸探针的研制奠定了基础.     

流行性乙型脑炎病毒NS4A蛋白基因的克隆及其在大肠埃希菌的表达

目的:克隆及表达流行性乙型脑炎病毒(JEV)NS4A蛋白基因.方法:JEV JaGAr-01株感染C6/36细胞,RT-PCR法获取JEV NS4A蛋白基因并采用ABI PRSMTM310 Genetic Analyzer进行DNA测序分析,PCR克隆方法将RT-PCR产物构建于原核表达载体PET-3a的BamHI与EcoRI酶切位点并命名为pNS4A,电泳进行限制性内切酶分析.结果:JEV NS4A蛋白基因大小为446 bp,DNA测序与发表的JEV JaGAr-01株相对应的DNA序列相符合,酶切后从重组质粒释出的插入子与JEV NS4A蛋白基因大小相一致.pNS4A在原核细胞内表达的特异蛋白分子量约16 kDa,与JEV NS4A蛋白理论计算相一致.结论:获取的JEV NS4A蛋白基因来自于JaGAr-01野生株,pNS4A在大肠埃希菌中表达的蛋白产物为JEV NS4A蛋白.     

不同途径接种乙脑毒prME蛋白编码基因DNA疫苗所致BALB/c鼠的细胞免疫

目的:研究经不同途径接种乙脑病毒prME蛋白编码基因DNA疫苗所诱导的细胞免疫。方法:将乙脑病毒prME蛋白编码基因DNA疫苗(命名为pJME)分别以肌注、滴鼻两种免疫方式免疫BALB/c鼠,流式细胞仪检测脾脏CD4 /CD8 T淋巴细胞亚群,乳酸脱氢酶释放实验测定脾脏特异性细胞毒性T淋巴细胞(CTL)活性。结果:pJME肌注组与滴鼻组免疫鼠CD4 T淋巴细胞百分比(30.91 0.72)%、(27.89 0.42)%,均高于灭活疫苗与载体肌注组(23.73 0.17)%、(23.78 0.21)%(P<0.05),肌注组显著高于滴鼻组(P<0.05);而CD8 T淋巴细胞百分比(14.62 0.25)%、(13.73 0.50)%,也均高于灭活疫苗和载体肌注组(10.80 0.39)%、(11.97 0.06)%(P<0.05),肌注组与滴鼻组无显著差异(P>0.05);两组有明显CTL效应(pJME肌注组29.1%,pJME滴鼻组17.1%),明显高于载体肌注组6.0%,pJME肌注组明显优于滴鼻组。结论:pJME以两种途径免疫鼠时可诱导明显细胞免疫反应。     

流行性乙型脑炎病毒E蛋白基因在大肠杆菌中的克隆与表达

目的;获得JEV E蛋白基因,构建重组表达载体并在E.coli中高效表达.方法:参照GeneBank中的日本乙型脑炎病毒SA14-14株序列设计一对特异性引物,采用RT-PCR法扩增E基因全长,克隆至PUCm-T载体,经测序证实后克隆至原核表达载体pET32a中,转化入BL21(DE3),IPTG诱导表达,对表达产物进行SDS-PAGE电泳分析.结果:限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明,扩增出了JEV E蛋白基因全长1500bp,成功构建了重组质粒pET-32a/JEV E,SDS-PAGE分析显示目的蛋白主要以包涵体的形式在大肠杆菌中获得表达,表达量占总菌体蛋白的35%.结论:在大肠杆菌中成功表达了JEV E蛋白,为ELISA早期诊断试剂盒的研制和E蛋白基因结构与功能的分析奠定了基础.     

日本脑炎病毒核心蛋白酵母双杂交诱饵载体构建及鉴定

目的 构建日本脑炎病毒核心蛋白(JEV C蛋白)基因的酵母双杂交诱饵载体,并检测其蛋白表达产物对酵母细胞的毒性及对酵母细胞内报告基因的自激活效应.方法 PCR扩增JEV C蛋白cDNA全基序并克隆入载体pGBKT7的EcoR1/BamHI酶切位点之间,酶切及测序鉴定;PE G/LiAc法将构建好的诱饵质粒pGBKT7-JC及载体pGBKT7分别转化人酵母菌株Y2H Gold感受态细胞,经酵母氨基酸缺陷培养及表型筛选检测其对酵母细胞毒性及自激活作用;用Western-blotting法分析酵母菌中诱饵蛋白BD-JC的表达.结果 重组质粒pGBKT7-JC经酶切鉴定及测序结果与Genebank公布的JEV SA14-14-2株中JEV C蛋白编码基序完全一致;成功转化质粒pGBKT7-JC及空载体pGBKT7的酵母菌株Y2H Gold感受态细胞均只在SD/-Trp、SD/-Trp/X培养基上生长,二者菌落数、菌落大小、分布无明显差异且不变蓝,且SD/-Trp液体培养基30℃过夜培养测OD600值均＞0.8,在SD/-TrpPMA培养基上均不生长;经Western-blotting法检测到酵母菌能表达分子量约为35KDa的特异性融合蛋白BD-JC.结论 成功构建了酵母双杂交诱饵表达质粒pCBKT7-JC;其诱饵蛋白BD-JC对酵母细胞无细胞毒性及自激活效应,为筛选及验证与pCBKT7-JC相互作用蛋白奠定实验基础.     

日本脑炎病毒核心蛋白酵母双杂交诱饵载体构建及鉴定

目的构建日本脑炎病毒核心蛋白（JEV C蛋白）基因的酵母双杂交诱饵载体,并检测其蛋白表达产物对酵母细胞的毒性及对酵母细胞内报告基因的自激活效应。方法 PCR扩增JEV C蛋白cDNA全基序并克隆入载体pGBKT7的EcoRI/BamHI酶切位点之间,酶切及测序鉴定;PEG/LiAc法将构建好的诱饵质粒pGBKT7-JC及载体pGBKT7分别转化入酵母菌株Y2H Gold感受态细胞,经酵母氨基酸缺陷培养及表型筛选检测其对酵母细胞毒性及自激活作用;用Western-blotting法分析酵母菌中诱饵蛋白BD-JC的表达。结果重组质粒pGBKT7-JC经酶切鉴定及测序结果与Genebank公布的JEV SA14-14-2株中JEV C蛋白编码基序完全一致;成功转化质粒pGBKT7-JC及空载体pGBKT7的酵母菌株Y2H Gold感受态细胞均只在SD/-Trp、SD/-Trp/X培养基上生长,二者菌落数、菌落大小、分布无明显差异且不变蓝,且SD/-Trp液体培养基30℃过夜培养测OD600值均〉0.8,在SD/-Trp/X/A培养基上均不生长;经Western-blotting法检测到酵母菌能表达分子量约为35KDa的特异性融合蛋白BD-JC。结论成功构建了酵母双杂交诱饵表达质粒pGBKT7-JC;其诱饵蛋白BD-JC对酵母细胞无细胞毒性及自激活效应,为筛选及验证与pGBKT7-JC相互作用蛋白奠定实验基础。     

二球悬铃木花粉主要变应原蛋白编码基因克隆的制备

目的:构建含二球悬铃木花粉主要变应原蛋白编码基因的重组质粒。方法:用PCR扩增二球悬铃木花粉主要变应原编码基因的cDNA序列,将扩增的目的片段用双酶切法与质粒pET32a连接,CaCl2法转入工程菌DH5α制备基因克隆。碱裂解法提取质粒DNA,测序鉴定目的片段存在。结果:构建了含二球悬铃木花粉主要变应原编码基因的重组质粒。结论:用天然二球悬铃木花粉可成功地制备含有其主要变应原编码基因的重组质粒,用于变应原蛋白诱导表达。     

乙型肝炎病毒X基因的克隆与鉴定

目的 构建乙型肝炎病毒X基因原核表达质粒pQE30-HBx,以进一步研究其基因产物的功能及致病机制.方法 以肝炎患者血清DNA为模板,用PCR方法扩增乙型肝炎病毒X基因,构建乙型肝炎病毒X基因原核表达质粒pQE30-HBx,经酶切电泳验证重组质粒的正确性,并对X基因进行测序鉴定.结果 PCR结果显示,扩增片断大小约465 bp,与预期相同.重组质粒经酶切电泳判断有目的片段插入,方向正确,经测序证实插入片段是乙型肝炎病毒X基因,编码框无误.结论 成功构建乙型肝炎病毒X基因重组质粒pQE30-HBx.     

GFP-HPVl8 E2删除突变体融合蛋白真核表达载体的构建与鉴定

目的 构建人乳头瘤病毒18型早期蛋白2(HPV18 E2)删除突变体N端(TAD)、C端(DBD)与增强型绿色荧光蛋白(GFP)融合蛋白的真核表达载体,为研究HPV18 E2基因与宫颈癌发生的关系提供实验基础.方法 用PCR扩增HPV18 E2 TAD、DBD基因序列,用EcoRⅠ和BamHⅠ双酶切pEGFP-Cl载体与TAD、DBD基因PCR产物,纯化回收后,用连接酶连接并转化大肠杆菌JM109,筛选阳性克隆,并对其进行双酶切和测序鉴定.结果 阳性克隆质粒经双酶切后,琼脂糖凝胶电泳可见4.7 kb、618 bp和243 bp附近的片段.DNA测序结果经与GenBank登录的序列做BLAST分析,显示重组质粒分别含有618 bp与243 bp的目的基因片段,无碱基错配和移码突变,读码框完全正确.结论 成功地构建了GFP-HPV18 E2删除突变体融合蛋白真核表达载体pEGFP-C1/TAD与pEGFP-C1/DBD.     

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.     

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…     

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.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.