Cyr61蛋白表达载体的构建及表达

目的：体外表达并纯化Cyr61蛋白片段。方法：提取乳腺癌组织总RNA，用一步法RT—PCR选择扩增编码Cyr61蛋白的cDNA片段，并将之克隆到融合蛋白表达载体pQE80L中，转染大肠杆菌Rosetta—gami^TM2。异丙基硫代半乳糖苷（IPTG）诱导表达融合蛋白，用亲和层析法纯化。纯化产物经聚丙烯酰胺凝胶电泳及蛋白质印迹分析鉴定。结果：克隆到编码Cyr61蛋白片段的cDNA片段，其大小为930bp。构建的表达质粒PQE80-Cyr61经限制性内切酶酶切和DNA测序证实为所需要的质粒。表达出相对分子质量分别为32kD的可溶性融合蛋白，经蛋白质印迹分析鉴定为Cyr61的融合蛋白。结论：本实验克隆了编码Cyr61蛋白片段的cDNA序列，并成功地获得了可溶性表达的融合目的蛋白，为进一步制备抗Cyr61蛋白的抗体和建立Cyr61蛋白定量检测方法创造了条件。     

人细胞核自身抗原精子蛋白的重组表达及多克隆抗体制备

目的:获得纯化的人细胞核自身抗原精子蛋白(hNASP)及其多克隆抗体,为其功能研究做准备。方法:提取人睾丸组织总RNA,用自行设计的引物,PCR扩增hNASP的一段序列,PCR产物经TA克隆后,通过BamHⅠ和HindⅢ双酶切克隆到pET-28 a(+)中。在E.coliBL21中,用异丙基-β-硫代半乳糖苷(IPTG)诱导表达H is融合蛋白。样品超声处理后,经镍离子亲和树脂进行亲和层析纯化。用纯化的重组蛋白免疫家兔获取多克隆抗体。结果:对表达重组蛋白的质粒进行DNA测序以及表达的重组蛋白经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,证实获取了目的蛋白。ELISA证实免疫家兔后获得高效价的抗体。结论:用上述原核生物表达的方法可以得到纯化的hNASP蛋白,用纯化的蛋白免疫家兔也能获得高效价的抗体。     

乙型肝炎病毒X抗原结合蛋白1的原核表达及亚细胞定位

目的应用酵母双杂交及生物信息学技术筛选乙型肝炎病毒X抗原结合蛋白1(HBXBP1)并进行克隆,探讨其在大肠埃希菌BL21中表达及亚细胞定位。方法应用反转录PCR(RT-PCR)技术,以HepG2细胞mRNA为模板,扩增获得HBXBP1基因片段,连接到pGEM-T载体,双酶切鉴定及测序正确后克隆至原核表达载体pET-32a(+)中,转化大肠埃希菌BL21,通过IPTG诱导表达以获得HBXBP1融合蛋白,并经SDS-PAGE及Western blot分析证实融合蛋白的特异性;构建HBXBP1基因的绿色荧光蛋白表达质粒pEGFP-C1-HBXBP1,转染HepG2细胞,24h后于荧光显微镜下观察表达蛋白的亚细胞定位。结果 RT-PCR扩增获得921bp的HBXBP1基因片段,插入原核表达载体pET-32a(+)中,转化大肠埃希菌BL21,经IPTG诱导成功获得约56kD的重组蛋白,经Western blot分析证实其具有良好的特异性;成功构建HBXBP1基因的绿色荧光蛋白表达质粒pEGFP-C1-HBXBP1,其表达蛋白定位于细胞质。结论 pET-32a(+)-HBXBP1原核表达载体在大肠埃希菌BL21中诱导表达HBXBP1融合蛋白;HBXBP1基因可表达HBXBP1蛋白且定位于细胞质,为研究HBXBP1蛋白的免疫原性和生物学特性奠定了一定的理论基础。     

人Eppin基因的原核表达及抗原性鉴定

目的 构建人附睾蛋白酶抑制蛋白(epididymal protease inhibitor,eppin)基因原核表达质粒,表达并纯化重组蛋白,并探讨其抗原性及应用价值.方法 以健康人睾丸组织cDNA为模板进行PCR扩增,将其克隆到原核表达载体pET-32a,并转化大肠埃希菌BL21(DE3),异丙基-β-D硫代半乳糖苷(IPTG)进行诱导表达,经Ni2+-NTA亲和层析柱纯化,并对纯化产物进行Western印迹鉴定.结果 重组质粒经双酶切分析和测序鉴定后证实构建成功;SDS-PAGE结果显示表达产物为相对分子质量36KD的融合蛋白,Western印迹检测纯化后蛋白显示特异性条带.结论 成功构建了pET-32a-Eppin重组质粒,并获得了具有抗原特异性的可溶性Eppin蛋白,为进一步研究其生物学活性及免疫性避孕效应奠定了基础.     

结核分枝杆菌抗原ESAT-6和CFP-10及rCFP10-ESAT6融合基因在大肠埃希菌中的表达及比较

目的 构建结核早期分泌抗原靶分子(ESAT-6)、培养滤液蛋白10(CFP-10)及重组CFP10-ESAT6(rCFP10-ESAT6)融合蛋白的原核表达载体,获得纯化蛋白并比较三者表达特点.方法 应用聚合酶链反应(PCR)技术,扩增ESAT-6、CFP-10基因片段,融合PCR技术扩增rCFP10-ESAT6片段,分别连接到pGEM-T载体,测序正确后插入原核表达载体pET-32a(+)或 pET-28a(+)中,转化大肠埃希菌BL21(DE3),IPTG诱导表达,亲和层析法纯化3种蛋白;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot分析验证目的蛋白的抗原特异性.结果 pET-32a(+)-ESAT6融合蛋白以包涵体形式表达,分子量为31 kD,目的蛋白约占菌体总蛋白的42%;pET-32a(+)-CFP10、pET-28a(+)-CFP10-ESAT6重组蛋白以可溶性形式表达,分子量分别为33 kD和28 kD,目的蛋白分别占菌体总蛋白的73%和48%;Western blot证实其均具有良好的抗原性.亲和层析法获得了纯度较高的重组蛋白.结论 利用大肠埃希菌BL21(DE3)成功表达了具有良好抗原性的ESAT-6、CFP-10融合蛋白及rCFP10-ESAT6重组蛋白,为深入研究其免疫原性和生物学特性奠定了基础.     

JC病毒Agnoprotein在大肠埃希菌中的表达及纯化

目的 构建JC病毒Agnoprotein基因的原核表达载体,表达纯化该蛋白.方法 PCR扩增患者脑脊液中JC病毒晚期调节蛋白Agnoprotein基因,测序正确后克隆入pET-32a(+)原核表达载体,诱导表达Agnoprotein蛋白并纯化.结果 pET-32a(+)-Agnoprotein表达出预期分子量大小的具有免疫活性的重组融合蛋白.结论 成功地表达纯化了Agnoprotein融合蛋白,为进一步研究其生物功能奠定了基础.     

人细胞核自身抗原精子蛋白的重组表达及克隆抗体制备

目的：获得纯化的人细胞核自身抗原精子蛋白（hNASP）及其多克隆抗体,为其功能研究做准备.方法：提取人睾丸组织总RNA,用自行设计的引物,PCR扩增hNASP的一段序列,PCR产物经TA克隆后,通过BamH Ⅰ和HindⅢ双酶切克隆到pET-28a（＋）中.在E.coli BL21中,用异丙基-β-硫代半乳糖苷（IPTG）诱导表达His融合蛋白.样品超声处理后,经镍离子亲和树脂进行亲和层析纯化.用纯化的重组蛋白免疫家兔获取多克隆抗体.结果：对表达重组蛋白的质粒进行DNA测序以及表达的重组蛋白经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析,证实获取了目的蛋白.ELISA证实免疫家兔后获得高效价的抗体.结论：用上述原核生物表达的方法可以得到纯化的hNASP蛋白,用纯化的蛋白免疫家兔也能获得高效价的抗体.     

人Tnfrsfl 1b-N端多狀的原核表达、纯化及其初步分析

目的 对原核表达和可溶性纯化后的Tnfrsf11b蛋白氨基端的24～106位肽段进行二级结构分析.方法 根据已经发表的人Tnfrsf11b氨基酸序列,利用BLAST程序进行同源性分析,确定所要构建的Tnfrsf11b N 端CRD功能结构域;将目的 基因片段克隆入表达载体 pGEX-6P-1并转化大肠杆菌BL21(DE3),于16.C、0.1 mmol/L IPTG 诱导25～30 h可获得可溶性融合蛋白,经过Glutathione SepharoseTM4 B 亲和层析纯化和 PreScission Protease 酶切,洗脱的蛋白样品经过Superdex75凝胶预装柱进一步纯化,最终获得可溶性、高纯度的蛋白;对目标蛋白进行圆二色谱分析.结果 获得重组质粒pCEX-6P-1-tnfrsf11b并在大肠杆菌中可溶性表达,经亲和层析、酶切、分子筛层析后获得了可溶的、高纯度的Tnfrsf11bN 端功能结构域蛋白并对其进行了圆二色谱分析,发现目标蛋白富含β-sheet.结论 高含量的β-sheet 更有利于蛋白质参与与相互作用,这对其功能的发挥有着重要意义;对此进行研究可以更好的理解这种相互作用,并为进一步研究Tnfrsf11bN端的结构和功能奠定了基础.     

FAM172A及其异构体重组蛋白对体外培养肝细胞增殖的影响

目的体外克隆人类基因FAM172A,构建其原核表达载体并诱导其重组蛋白的表达,制备兔抗FAM172A重组蛋白的多克隆抗体,观察其在不同细胞系的表达情况。观察FAM172A-1和FAM172A-3蛋白对L02细胞增殖的影响。方法利用反转录聚合酶链反应（RT-PCR）及PCR技术,构建原核表达质粒pET-32a（＋）-FAM172A-1和pET-32a（＋）-FAM172A-3。诱导该基因不同异构体重组蛋白的表达,并通过蛋白质免疫印迹（Western blot）技术进行鉴定。纯化后的重组蛋白FAM172A-1和FAM172A-3与肝细胞L02细胞共孵育,观察不同浓度的重组蛋白对细胞增殖的影响。利用纯化后的FAM172A（异构体3）重组蛋白免疫大耳白兔,获得抗FAM172A-3蛋白的多克隆抗体,利用酶联免疫吸附法（ELISA）以及Western blot技术对获得的多克隆抗体进行效价分析及特异性检测。结果成功扩增获得FAM172A（异构体1、3）的基因片段,测序结果与GenBank已公开的基因序列一致;成功表达FAM172A（异构体1、3）的重组蛋白,经过Western blot鉴定正确;纯化后的重组蛋白FAM172A （异构体1、3）与L02细胞共孵育结果发现,两者对L02细胞在一定浓度范围内均有促进细胞增殖的作用。所制备的兔抗人FAM172A-3重组蛋白的多克隆抗体,ELISA检测显示其效价可达1︰1280000, Western blot检测证实该多克隆抗体的特异性良好;Western blot分析显示,该蛋白在肝脏间质细胞和实质细胞均有一定程度的表达。结论 FAM172A蛋白在肝实质细胞及肝间质细胞均表达,且其重组蛋白可以促进L02细胞增殖,推测该基因可能与肝细胞损伤、肝纤维化以及肝细胞再生等发生机制有关。     

轮状病毒外壳蛋白VP4在大肠埃希菌中的表达

目的 克隆表达轮状病毒融合素蛋白VP4.方法 以轮状病毒SA11株细胞培养物提取总RNA为模板,经RT-PCR获得VP4全长基因片段cDNA,将其重组于pET-30a(+)表达载体,转化大肠埃希菌BL21(plysS),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经SDS-PAGE和Western blot鉴定,并用镍柱纯化.结果 重组载体所含目的基因片段的序列与文献公布序列相符,IPTG诱导表达的目的蛋白主要以包涵体形式存在;SDS-PAGE检测表达产物与目的蛋白大小一致,为88 kD,Western blot检测发现目的蛋白所带组氨酸标签可与标签抗体反应;镍柱纯化获得RV SA11 VP4融合素蛋白,纯度达90%.结论 构建了表达载体pET-30a(+)-VP4,经诱导表达、纯化,获得了RV融合素蛋白VP4.     

Perioperative and long-term morbidity and mortality after above-knee and below-knee amputations in diabetics and nondiabetics

We performed a retrospective review of a vascular surgery quality assurance database to evaluate the perioperative and long-term morbidity and mortality of above-knee amputations (AKA, n = 234) and below-knee amputations (BKA, n = 720) and to examine the effect of diabetes mellitus (DM) (181 of AKA and 606 of BKA patients). All patients in the database who had AKA or BKA from 1990 to May 2001 were included in the study. Perioperative 30-day cardiac morbidity and mortality and 3-yr and 10-yr mortality after AKA or BKA were assessed. The effect of DM on 30-day cardiac outcome was assessed by multivariate logistic regression and the effect on long-term survival was assessed by Cox regression analysis. The perioperative cardiac event rate (cardiac death or nonfatal myocardial infarction) was at least 6.8% after AKA and at most 3.6% after BKA. Median survival was significantly less after AKA (20 mo) than BKA (52 mo) (P < 0.001). DM was not a significant predictor of perioperative 30-day mortality (odds ratio, 0.76 [0.39-1.49]; P = 0.43) or 3-yr survival (Hazard ratio, 1.03 [0.86-1.24]; P = 0.72) but predicted 10-yr mortality (Hazard ratio, 1.34 [1.04-1.73]; P = 0.026). Significant predictors of the 30-day perioperative mortality were the site of amputation (odds ratio, 4.35 [2.56-7.14]; P < 0.001) and history of renal insufficiency (odds ratio, 2.15 [1.13-4.08]; P = 0.019). AKA should be triaged as a high-risk surgery while BKA is an intermediate-risk surgery. Long-term survival after AKA or BKA is poor, regardless of the presence of DM.     

Postoperative nausea and vomiting and opioid-induced nausea and vomiting: guidelines for prevention and treatment

Postoperative nausea and vomiting (PONV) causes patient discomfort, lowers patient satisfaction, and increases care requirements. Opioid-induced nausea and vomiting (OINV) may also occur if opioids are used to treat postoperative pain. These guidelines aim to provide recommendations for the prevention and treatment of both problems. A working group was established in accordance with the charter of the Sociedad Espa?ola de Anestesiología y Reanimación. The group undertook the critical appraisal of articles relevant to the management of PONV and OINV in adults and children early and late in the perioperative period. Discussions led to recommendations, summarized as follows: 1) Risk for PONV should be assessed in all patients undergoing surgery; 2 easy-to-use scales are useful for risk assessment: the Apfel scale for adults and the Eberhart scale for children. 2) Measures to reduce baseline risk should be used for adults at moderate or high risk and all children. 3) Pharmacologic prophylaxis with 1 drug is useful for patients at low risk (Apfel or Eberhart 1) who are to receive general anesthesia; patients with higher levels of risk should receive prophylaxis with 2 or more drugs and baseline risk should be reduced (multimodal approach). 4) Dexamethasone, droperidol, and ondansetron (or other setrons) have similar levels of efficacy; drug choice should be made based on individual patient factors. 5) The drug prescribed for treating PONV should preferably be different from the one used for prophylaxis; ondansetron is the most effective drug for treating PONV. 6) Risk for PONV should be assessed before discharge after outpatient surgery or on the ward for hospitalized patients; there is no evidence that late preventive strategies are effective. 7) The drug of choice for preventing OINV is droperidol.     

Body composition assessment and methodology in nonathletic and athletic adolescent and adult males and females

The purpose of this review is to outline methodology for assessing body composition utilizing anthropometric and densitometric techniques. The objective of body composition assessment is to measure body fat and lean body mass. The quantity of these components varies due to growth, physical activity, dietary regimens, and aging. Anthropometric techniques incorporate selected skinfolds, circumferences, skeletal widths, or other variables to estimate body composition within k2.0-4.0%. These techniques are adequate for field testing of groups or individuals, but are population specific. Densitometry measures body volume irrespective of physique, sex, or age. This laboratory technique estimates body composition within 1.0-2.0%, is more difficult to administer, but is not population specific. Some limitation exists with any present technique due to biological variability and incomplete research of reference body composition in children, females, and the aged. J Orthop Sports Phys Ther 1984;5(6):336-347.