结核分枝杆菌Rv0901基因原核表达质粒的构建及其表达

目的　为研究结核分枝杆菌基因Rv0 90 1的功能提供材料。方法　PCR扩增Rv0 90 1基因编码序列 ,定向克隆入融合蛋白原核表达载体pGEX 1λT获得重组表达质粒 ,转化大肠杆菌后用IPTG进行诱导表达 ,通过SDS PAGE、GST 纯化试剂盒鉴定并纯化表达产物。结果　从结核杆菌H37Rv株基因组DNA中扩增出Rv0 90 1基因 ;成功构建了融合表达质粒pGEX Rv0 90 1;重组质粒经IPTG诱导后能在大肠杆菌中稳定表达 4 5kDa的GST Rv0 90 1融合蛋白。结论　本实验成功表达并纯化GST Rv0 90 1融合蛋白 ,为Rv0 90 1基因功能的研究打下了基础。     

人氨肽酶N基因克隆和原核表达

目的：构建人氨肽酶N(APN)-谷胱甘肽巯基转移酶(GST)融合蛋白(APN-GST)，并进行原核表达。方法：根据人APN mRNA序列设计合成特异性引物，从人肝组织中提取总RNA并反转录成cDNA第一链，RT-PCR扩增人APN基因，并插入融合蛋白原核表达载体pGEX-4T，转化宿主菌BL21(DE3)细胞，异丙基锍基半乳糖(IPTG)诱导表达APN蛋白。包涵体经尿素变性、重折叠和亲和层析纯化。结果：重组质粒测序和酶切结果显示：APN基因已正确插入pGEX-4T，重组蛋白及纯化产物SDS-PAGE在135000处有一条明显的蛋白表达条带。结论：人APN基因已被成功地克隆、表达和纯化。     

人重组蛋白P311的原核表达纯化与鉴定

目的 原核表达人增生性瘢痕新候选相关蛋白P311,并对该重组蛋白进行纯化、鉴定.方法 通过PCR方法扩增人P311基因,利用BamH Ⅰ和Xho Ⅰ酶切位点将其克隆至谷胱甘肽-S-转移酶(glutathione-s-transferase,GST)融合蛋白表达载体pGEX-4T-1中,转化大肠杆菌BL21(DE3)后,经异丙基硫代-β-D-半乳糖苷(isopropyl-β-D-5-thiogalactoside,IPTG)诱导表达,用SDS-PAGE、Western blotting等方法鉴定表达产物,并用谷胱甘肽-琼脂糖小珠亲和纯化表达的GST-P311蛋白.结果 重组载体pGEX-4T-P311经酶切与测序鉴定证实构建成功.导入大肠杆菌进行表达,表达产物相对分子量为34KD左右,与预期值相符.该条带经免疫印迹检测鉴定为GST抗体阳性,获得了纯化的GST-P311蛋白.结论 构建了pGEX-4T-P311原核表达载体,并在大肠杆菌BL21(DE3)中成功表达,亲和纯化获得较高纯度的GST融合蛋白,为下一步继续研究P311的生物学功能奠定了基础.     

受精抗原-1基因的克隆和GST融合表达

目的：克隆和原核表达小鼠受精抗原-1(FA-1)基因。方法：自行设计引物，用RT-PCR从小鼠睾丸组织总RNA扩增FA-1基因的编码序列。将扩增产物克隆至GST融合表达载体pGEX-2T。以IPTG诱导GST融合FA-1的表达。结果：凝胶电泳显示PCR扩增产物的分子量与预期的508bp-致。重组质粒经BamHI／EcoR I双酶切后产生一个约500bp的片段。对重组质粒的序列测定表明，插入片段的序列与发表的FA-1基因编码序列相符。在IPTG的诱导下，BL21重组菌高效表达出一个分子量与43kDa相近的产物(预期44.2kDa)。该产物可通过GST融合蛋白纯化试剂盒得到纯化。结论：小鼠FA-1编码序列已被成功地克隆至GST融合表达载体pGEX-2T。     

迁移侵袭抑制蛋白MIIP的GST融合蛋白表达及生物学活性鉴定

目的构建人MIIP蛋白与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体并进行表达及生物学活性鉴定。方法 RT-PCR扩增得到人MIIP基因全长编码序列,采用重组DNA技术将其克隆至原核表达载体pGEX-4T-1,构建获得重组原核表达载体pGEX-4T-1-MIIP,经酶切鉴定及测序验证完全正确后,将其转化至大肠杆菌BL21细胞中,用异丙基硫代-β-D半乳糖苷(IPTG)诱导表达,经纯化获得目的蛋白,用Western blot鉴定所得融合蛋白。结果构建了pGEX-4T-1-MIIP原核表达载体,在大肠杆菌中表达获得了GST-MIIP融合蛋白,经Glutathione Agarose纯化和Western blot检测,证实所得GST-MIIP融合蛋白具有生物学活性。结论成功获得了有生物学活性的GST-MIIP融合蛋白。     

都柏林沙门氏菌metk基因克隆及原核表达

目的获取都柏林沙门氏菌metk基因编码s-腺苷甲硫氨酸合成酶His-tag融合蛋白,为基因工程生产s-腺苷甲硫氨酸创造条件。方法利用都柏林沙门氏菌metk基因特异引物,以都柏林沙门氏菌临床分离株的基因组DNA为模板,利用PCR技术扩增得到该基因编码区,并构建原核表达重组质粒载体pET30a-metk,经酶切鉴定后转化大肠杆菌BL21（DE3）,IPTG诱导融合蛋白表达,Ni^2＋-NTA柱纯化重组蛋白。结果经IPTG24℃诱导6h后,12%SDS-PAGE电泳检测表明诱导表达的融合蛋白占细菌总蛋白高达40%以上,重组蛋白分子量约为58.8kDa。结论成功构建His-METK融合蛋白原核表达载体,并高效表达、纯化、复性,为进一步利用该蛋白奠定了基础。     

多功能转录因子CTCF重组质粒构建及其表达鉴定

目的 构建人CTCF cDNA全长及N端、Zn指、C端3个片段的原核融合表达载体,纯化融合蛋白并进行鉴定.方法 以编码CTCF全长序列的质粒为PCR模板,分别构建GST融合表达重组质粒pGEX-4T-2-CTCF,pGEX-4T-2-CTCF-N,pGEX-4T-2-CTCF-Zn,pGEX-4T-2-CTCF-C,转化大肠杆菌BL21,酶切、测序鉴定;优化IPTG诱导表达条件,并对亲和层析纯化的GST融合蛋白CTCF,CTCF-N,CTCF-Zn,CTCF进行Far-West-ern blot鉴定.结果 重组质粒经双酶切和测序鉴定证实构建成功.GST融合蛋白CTCF,CTCF-N,CTCF-Zn,CTCF-C均在大肠杆菌中成功诱导表达,融合蛋白经亲和层析纯化获得纯化蛋白.各纯化蛋白经SDS-PAGE和Far-Western blot鉴定,均为诱导表达蛋白质.结论 成功构建了GST融合表达CTCF,CTCF-N,CTCF-Zn,CTCF-C的重组质粒,对融合蛋白表达条件进行了优化.获得了高效表达的GST融合蛋白.     

ABH1融合蛋白表达载体的构建及表达纯化

目的：构建ABH1重组蛋白原核表达载体及建立ABH1蛋白纯化技术。方法：应用PCR技术从人的cDNA文库扩增出ABH1基因片段,并加入限制性内切酶位点和终止子,进而将其插入到原核表达质粒pET-15b中,构建ABH1/15b原核表达克隆,最后用IPTG诱导其在大肠杆菌BL21(DE3)中表达,用镍柱亲和纯化,离子交换层...     

重组原核表达载体pGEX-4T-1-MAGE-1的构建及表达

目的:构建人肿瘤相关抗原MAGE-1基因原核表达载体.方法:从人肝细胞性肝癌组织中提取总RNA,RT-PCR扩增出MAGE-1基因.酶切鉴定后,将其插入pGEM-T载体,再克隆至原核表达载体pGEX-4T-1中,构建重组表达质粒pGEX-4T-1-MAGE-1.将重组质粒转化大肠杆菌BL21(DE3),经1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导4 h,SDS-PAGE及凝胶密度扫描分析目的蛋白的表达情况.结果:RT-PCR扩增出长度为927 bp的MAGE-1基因.重组表达质粒pGEX-4T-1-MAGE-1转化大肠杆菌BL21(DE3)后经诱导表达,目的蛋白约57 000,占菌体总蛋白的23%.结论:成功构建出重组原核表达载体pGEX-4T-1-MAGE-1,该载体在大肠杆菌BL21(DE3)中高效表达.     

人膜联蛋白Ⅴ基因的克隆及原核表达

目的构建人膜联蛋白ⅤcDNA的原核表达载体并诱导其表达。方法利用RT-PCR技术从人胎盘组织总RNA扩增膜联蛋白Ⅴ基因的编码序列,将扩增产物克隆至GST融合表达载体pGEX-2T,以IPTG诱导GST融合人膜联蛋白Ⅴ的表达。结果凝胶电泳显示人胎盘组织PCR扩增产物的分子质量大小与目的片段大小相符。对重组质粒分析表明,插入片段的序列与发表的人膜联蛋白Ⅴ基因编码序列一致。在IPTG的诱导下,BL21重组菌高效表达出一个相对分子质量约为60000的产物。结论人膜联蛋白Ⅴ编码序列已被克隆至GST融合表达载体pGEX-2T。     

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…     

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.     

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.     

Dr.Zhang Ren's Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

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.     

猪肺磷脂注射液联合经鼻持续气道正压通气对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性的影响

目的 探讨猪肺磷脂注射液联合经鼻持续气道正压通气(NCPAP)对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性(CK-MB)的影响.方法 选取呼吸衰竭早产儿80例,分为观察组和对照组各40例.对照组采用NCPAP给氧治疗,观察组给予NCPAP给氧联合猪肺磷脂气管内给药.观察两组患儿治疗前及治疗12h、24 h后PaO2、PaCO2、血氧饱和度(SaO2)、pH的变化情况,检测治疗前及治疗5d后血清CK-MB水平;评估两组患儿的临床治疗效果.结果 两组患儿PaO2、PaCO2、SaO2、pH比较,差异均有统计学意义(P＜0.05),其中观察组治疗后的PaO2、SaO2、pH均高于对照组,PaCO2则低于对照组.两组的PaO2、SaO2、pH均随观察时间延长而升高(P＜0.05),PaCO2均随观察时间的延长而降低(P＜0.05).观察组治疗有效率为87.5％,显著高于对照组的70.0％ (P ＜0.05).治疗5d后两组患儿血清CK-MB水平均较前降低(P＜0.05),且观察组明显低于对照组(P＜0.05).结论 猪肺磷脂注射液气管内给药联合NCPAP可以显著降低呼吸衰竭早产儿CK-MB的含量,提高治疗有效率,起到很好的呼吸循环支持作用.