乙型肝炎病毒前-X蛋白在大肠埃希菌中的表达及多克隆抗体的制备

目的在大肠埃希菌中表达乙型肝炎病毒(HBV)前-X蛋白,纯化、鉴定并制备多克隆抗体。方法通过PCR技术获得HBV前-X编码基因片段,克隆至载体pET32a( ),构建原核表达重组质粒,转化至大肠埃希菌中诱导表达,表达产物进行Ni 亲和层析纯化,经Western blotting证实该蛋白的免疫原性,然后免疫新西兰兔获得多克隆抗体并经ELISA实验测定效价,经Western blotting证实其免疫反应特异性。结果成功构建原核表达载体并在大肠埃希菌中高效表达,表达产物主要以包涵体形式存在,融合蛋白的相对分子量约为25kD,Western blotting结果表明目的蛋白具有特异性的免疫反应识别。成功获得前-X蛋白的多克隆抗体,ELISA检测抗体效价为>1:128000,Western blotting实验证实获得的抗体能发生特异性免疫反应。结论成功完成了HBV前-X蛋白的体外表达及多克隆抗体制备,为检测前-X蛋白在乙型肝炎患者体内的表达及进一步研究其生物学特性奠定了实验基础。     

乙型肝炎病毒e抗原结合蛋白1的克隆及原核表达

目的 对应用酵母双杂交技术及生物信息学技术筛选得到的乙型肝炎病毒e抗原结合蛋白1(HBEBP1)进行克隆,并诱导其在大肠埃希菌BL21中的表达.方法 应用反转录PCR(RT-PCR)技术,以HepG2细胞mRNA为模板,扩增获得HBEBP1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,IPTG诱导表达以获得HBEBP1融合蛋白,并通过SDS-PAGE及Western blot证实融合蛋白的特异性.结果 RT-PCR扩增获得大小为372bp的HBEBP1基因片段,插入至原核表达载体pET-32a( )中,转化大肠埃希菌BL21,经IPTG诱导后,成功获得大小约为33kD的重组蛋白,Western blot证实其具有良好的特异性.结论 构建了pET-32a( )-HBEBP1原核表达载体,并在大肠埃希菌BL21中诱导表达了HBEBP1融合蛋白,为研究HBEBP1蛋白的免疫原性和生物学特性奠定了基础.     

丙型肝炎病毒F蛋白反式激活基因2的表达纯化及多克隆抗体制备

目的构建丙型肝炎病毒(HCV)F蛋白反式激活基因2(FTP2)的原核表达载体并诱导其表达,制备多克隆抗体并探讨其在临床病理组织中表达的意义。方法通过PCR获得HCVFTP2基因,将其克隆至原核表达载体pET32a( )上,构建重组表达载体,在大肠埃希菌BL21中诱导表达。表达产物经SDS-PAGE及Western blotting验证后进行大量表达并纯化。将纯化的重组蛋白免疫家兔获得多克隆抗体,并将此抗体作为诊断试剂观察其在正常肝组织和临床病理组织中的表达情况。结果以pET32a( )-FTP2表达载体转化BL21菌后,经IPTG诱导,成功获得大量重组蛋白,分子量约为25kD。将此重组蛋白免疫新西兰兔获得多克隆抗体,经抗体间接ELISA检测证明,多克隆抗体效价>1:128000,免疫组化显示多克隆抗体能够与丙型肝炎、肝硬化、肝细胞癌等临床病理组织产生FTP2抗原反应。结论成功表达了HCV的FTP2蛋白并制备了其多克隆抗体,证实FTP2与丙型肝炎、肝硬化的发病机制密切相关。     

肿瘤相关抗原MART-1蛋白原核表达载体的构建、表达纯化和活性鉴定

目的构建编码肿瘤相关抗原MART-1融合基因的原核表达载体,在大肠埃希菌中表达His-MART-1融合蛋白,并对蛋白进行纯化和免疫原性鉴定。方法采用PCR法扩增编码MART-1的基因片段,将该基因片段克隆到含有His标签的pET-28b原核表达载体上,重组质粒经双酶切、PCR及测序鉴定正确后转化大肠埃希菌,IPTG诱导融合蛋白表达,用镍离子亲和层析法纯化融合蛋白并进行脱盐,SDS-PAGE电泳和Western blotting法鉴定。通过ELISA法检测经树突状细胞提呈后的His-MART-1融合蛋白对特异性CD4+T细胞的刺激作用。结果重组质粒经双酶切、PCR和测序鉴定证明载体构建成功。His-MART-1融合蛋白经表达纯化后,分子量约13kD,与预期值相符。Western blotting证实该融合蛋白可与抗His单克隆抗体发生特异性结合。His-MART-1融合蛋白经树突状细胞提呈后能刺激MART-1特异性CD4+T细胞分泌IFN-γ。结论成功构建了编码MART-1基因原核表达载体pET-28b-MART-1,表达及纯化获得该融合蛋白,并证实其具有良好的免疫原性。     

丙型肝炎病毒NS5A反式激活基因1的原核表达及多克隆抗体制备

目的构建丙型肝炎病毒非结构蛋白NS5A的反式激活蛋白1(NS5ATP1)基因的原核表达载体并诱导其表达,纯化表达的融合蛋白并制备多克隆抗体。方法从pGBKT7-NS5ATP1质粒上切取NS5ATP1基因,克隆入pET32a( )质粒,构建pET32a( )-NS5ATP1表达载体,IPTG诱导融合蛋白表达。亲和镍柱层析纯化该融合蛋白后,免疫新西兰兔,获得多克隆抗体,ELISA法检测多抗效价。结果以pET32a( )-NS5ATP1表达载体分别转化DH5α、BL21菌后,经IPTG诱导,成功获得分子量为56kD左右的重组蛋白。SDS-PAGE分析显示,IPTG诱导4.5h时重组蛋白的表达量最高。Western blot证实其具有良好的抗原性。用该蛋白成功免疫新西兰白兔,获得了该蛋白的多克隆抗体,ELISA检测其效价>1∶512000。结论成功构建原核表达载体pET32a( )-NS5ATP1,表达纯化NS5ATP1融合蛋白并制备了该蛋白的多克隆抗体,为进一步的研究奠定了基础。     

人PD-L1胞外区基因的克隆、原核表达及抗体制备

目的 克隆人PD-L1胞外区基因并进行原核表达及相应蛋白的抗体制备,为进一步研究PD-1/PD-L1通路及针对该通路进行疾病治疗和药物筛选奠定基础.方法 用PCR方法扩增编码人PD-L1胞外区的基因序列,克隆到原核表达载体pET28a(+)中并进行表达,用His抗体为一抗进行Western blotting鉴定并验证结合活性.用纯化的hPD-L1胞外区蛋白作为抗原免疫日本大耳白兔,制备多克隆抗体.通过酶联免疫吸附实验(ELISA)和流式法检测抗体滴度及其特异性.结果 原核表达质粒pET28a(+)-hPD-L1成功构建,并可在大肠埃希菌BL21(DE3)中诱导高效表达,得到的hPD-L1胞外区蛋白经SDS-PAGE和Western blotting鉴定正确.用纯化蛋白免疫日本大耳白兔,制备的多克隆抗体具有较强的免疫结合活性,抗体滴度可达105.结论 鉴定和纯化了原核表达的hPD-L1蛋白,制备的相应多克隆抗体能够检测真核细胞表达的hPD-L1蛋白,为进一步研究PD-1/PD-L1生物学活性奠定了实验基础.     

小鼠附睾分泌蛋白Spink12的原核表达与纯化

目的原核表达带有His标签的小鼠附睾蛋白Spink12,对表达产物进行鉴定和纯化,以研究Spink12的免疫避孕效果。方法提取小鼠附睾组织总RNA,RT-PCR扩增不含信号肽的Spink12蛋白编码序列,以NdeⅠ和EcoRⅠ酶切后,将其连入原核表达载体pET28(a);经酶切和测序鉴定正确的重组质粒pET28(a)-Spink12,转化大肠埃希菌BL21感受态细胞。融合蛋白经IPTG诱导表达,产物经SDS-PAGE电泳和Western blotting免疫印迹鉴定。用Ni-NTA在非变性条件下利用不同的咪唑梯度浓度纯化融合蛋白6×His-Spink12。结果酶切和测序结果显示成功构建出原核表达载体pET28(a)-Spink12。重组质粒转化大肠埃希菌BL21并经IPTG诱导,SDS-PAGE电泳显示表达出约9kD的融合蛋白6×His-Spink12,与理论分子量相符。融合蛋白的表达量占菌体蛋白总量的23.5%。用抗His单克隆抗体进行Western blotting免疫印迹鉴定,检测到同样大小的条带。经纯化获得较高纯度的融合蛋白6×His-Spink12,纯度达91.1%。结论成功表达并纯化了融合蛋白6×...     

人前列腺干细胞抗原的原核表达、纯化及鉴定

目的 构建包含人前列腺干细胞抗原(PSCA)的原核表达质粒,并进行诱导表达、蛋白纯化及鉴定.方法 通过PCR扩增人PSCA基因片段,与过渡载体pGEM-T Easy连接后进行测序鉴定,鉴定正确后,将PSCA基因片段酶切并插入含有谷胱甘肽s-转移本科GST标签的原核表达载体pET42a,得到重组质粒pET42a-PSCA.将pET42a-PSCA转化大肠埃希菌BL21(DE3),在IPTG诱导下表达GST-PSCA融合蛋白,用Glutathione-Sepharose 4B柱对融合蛋白进行纯化,纯化产物进行SDS-PAGE及Western blotting鉴定.结果 PSCA基因的PCR产物大小与预期相符,测序结果与GenBank 中的PSCA序列一致.pET42a-PSCA原核表达质粒构建正确,SDS-PAGE及Western blotting检测显示融合蛋白诱导表达成功,分子量大小约为43kD.结论 成功构建了人PSCA的原核表达质粒,能在BL21菌株中表达,且纯化后得到较高纯度的GST-PSCA融合蛋白,为后续抗前列腺癌基因疫苗研究奠定了基础.     

丙型肝炎病毒NS5A反式激活蛋白7的重组表达及生物信息学分析

目的 构建丙型肝炎病毒NS5A反式激活蛋白7(NS5ATP7)的原核表达载体,诱导其在大肠埃希菌中的表达,并初步探讨其结构与功能.方法 应用逆转录PCR(RT-PCR)技术,以HepG2细胞mRNA为模板,扩增获得NS5ATP7基因片段,连接到pGEM-T载体,酶切鉴定及测序正确后插入至原核表达载体pET-32a( )中,转化大肠埃希菌BL21,IPTG诱导以获得NS5ATP7融合蛋白的表达,SDS-PAGE、Western blot免疫印迹分析和证实该融合蛋白表达的特异性,并应用生物信息学方法对该融合蛋白的结构和功能进行预测.结果 利用RT-PCR扩增获得大小为891bp的NS5ATP7基因片段,插入pET-32a( )表达载体,转化BL21宿主菌,经IPTG诱导,成功获得了大小为48kD的目的蛋白,Western blot进一步证实了该蛋白具有特异性免疫反应识别.生物信息学预测结果显示此蛋白富含螺旋结构,为非跨膜蛋白,无信号肽.结论 利用大肠埃希菌BL21成功表达了NS5ATP7融合蛋白,结合生物信息学分析结果,为研究NS5ATP7蛋白的免疫原性和生物学特性奠定了基础.     

乙型肝炎病毒DNA聚合酶反式调节人类新基因HBVDNAPTP1的克隆及原核表达与组织表达分析

目的 克隆应用抑制性消减杂交(SSH)技术及生物信息学技术筛选得到的乙型肝炎病毒(HBV)DNA聚合酶反式调节新型靶基因HBVDNAPTP1,构建HBVDNAPTP1基因的原核表达载体,诱导其在大肠埃希菌中表达,并进一步分析HBVDNAPTP1在组织中的分布表达水平.方法 应用RT-PCR技术扩增获得HBVDNAPTP1基因片段,测序正确后插入至原核表达载体pET-32a( )中,转化BL21(DE3)宿主菌,IPTG诱导以获得HBVDNAPTP1融合蛋白的表达,利用Western blot证实表达蛋白的特异性.应用UniGene数据库对HBVDNAPTP1基因的染色体中定位及组织分布表达水平进行分析.结果 RT-PCR扩增获得HBVDNAPTP1基因片段,插入pET-32a( )表达载体,转化BL21(DE3)受体菌,经IPTG诱导获得了HBVDNAPTP1重组蛋白的表达,Western blot证实了表达的重组蛋白的特异性.HBVDNAPTP1在多数组织中低表达,仅在脑垂体腺、扁桃体、舌、胸腺、气管与脐带中无表达.结论 成功构建了HBVDNAPTP1基因的原核表达载体,利用大肠埃希菌原核表达系统获得了重组蛋白的表达,并初步了解了HBVDNAPTP1基因的染色体定位与组织表达水平.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

Prevalence and severity of hip and groin pain in sub‐elite male football: a cross‐sectional cohort study of 695 players

The purpose of this study was twofold: (a) to investigate the prevalence of hip and groin pain in sub‐elite male adult football in Denmark and (b) to explore the association between prevalence and duration of hip and groin pain in the previous season with the Copenhagen Hip and Groin Outcome Score (HAGOS) in the beginning of the new season. In total 695 respondents from 40 teams (Division 1–4) were included. Players completed in the beginning of the new season (July–Sept 2011) a self‐reported paper questionnaire on hip and/or groin pain during the previous season and HAGOS. In total 49% (95% CI: 45–52%) reported hip and/or groin pain during the previous season. Of these, 31% (95% CI: 26–36%) reported pain for >6 weeks. Players with the longest duration of pain during the previous season had the lowest HAGOS scores, when assessed at the beginning of the new season, P < 0.001. This study documents that half of sub‐elite male adult football players report pain in the hip and/or groin during a football season. The football players with the longest duration of pain in previous season displayed the lowest HAGOS scores in the beginning of the new season.     

Bronchial stump fistula :treatment with covered retrievable hinged metallic stents-preliminary clinical experience

Objective To evaluate the preliminaily clinical efficacy and retrievability of a retrievable hinged covered metallic stent in the treatment of the bronchial stump fistula （BSF）. Methods Between April 2003 and March 2005, 8 patients with bronchial stump fistula after pneumonectomy or lobectomy were treated with two types （A and B） of retrievable hinged covered metallic stents. Type A stent was placed in 6 patients and type B in 2 under fluoroscopic guidance. The stent was removed with a retrieval set when BSF was healed or complications occurred. Results Stent placement in the bronchial tree was technically successful in all patients, without procedure-related complications. Immediate closure of the BSF was achieved in all patients after the procedure. Stents were removed from all patients but one. Removal of the stents was difficult in two patients due to tissue hyperplasia. Patients were followed up for 6 - 21 months. Placement of the stents remained stable in all patients except one due to severe cough. Permanent closure of BSF was achieved in 7 （87.5%） of 8 patients. Conclusion Use of a retrievable hinged covered expandable metallic stent is a simple, safe, and effective procedure for closure of the BSF. Retrieval of the stent seems to be feasible. （J Intervent Radiol, 2007, 16： 253-257）