在大肠杆菌中以非融合蛋白形式高效表达丙型肝炎核心蛋白

应用聚合酶链反应（ＰＣＲ）技术，扩增出完整的丙型肝炎病毒核心蛋白基因，将其克隆于表达载体ｐＢＶ２２０ ＰＲＰＬ启动子的下游，构建了重组质粒ｐＢＶ－ＨＣＶ；转化大肠杆菌后，以非融合蛋白方式表达了丙型肝炎病毒核心蛋白。ＳＤＳ－ＰＡＧＥ电泳显示，此蛋白的分子量约为２０ｋＤａ，表达量约占菌体蛋白的１１％；Ｗｅｓｔｅｒｎ印迹实验证明，此蛋白能与慢性丙型肝炎患者血清发生特异反应。序列分析结果表明，克隆于ｐＢＶ     

重组人IL-8基因的诱导表达及表达产生物生物学活性的初步鉴定

本文将通过序列分析证实的ＩＬ－８基因亚克隆到表达载体Ｐ＾ＢＶ２２０Ｋ ，ＳＱＣ ＶＦＨＰＤＮ Ｂ ＴＧＪ ＸＥＧ ＧＥ ＤＰ ＲＦＭ ＯＵＧ。     

利用T7和PR双启动子的新型表达载体的构建及其应用

构建具有λＰＲ与Ｔ７双启动动子的新型原核表达载体ｐＤＯＧ，以便使用同一载体研究不同诱导条件下，重组蛋白在大肠杆菌中表达，降解以及折叠的情况，从而选择最佳的诱导条件。方法采用ｐＢＶ２２０与ＰＥＴ－３表达载体作为原始材料，将ＰＥＴ－３上包括Ｔ７启动子与Ｔ７ｇ－１０释译起始序列的一段序列克隆到     

粒细胞巨噬细胞集落刺激因子重组质粒的构建及其在大肠杆菌中的表达

本研究通过反转录ＰＣＲ得到人ＧＭ-ＣＳＦ基因片段后，通过重组插入ｐＢＶ２２０载体质粒的ＥｃｏＲⅠ、ＢａｍＨⅠ之间，构建了重组表达质粒ｐｈＧＭ９１。借助计算机分析，优化了构建质粒的转译起始区，采取定点诱变以消除ＳＤ序列和ＡＴＧ区的强二级结构，提高了表达水平。将ＧＭ-ＣＳＦ基因进行序列测定，与文献报导完全一致，所对应的氨基酸序列与天然氨基酸序列一致。将带有表达质粒ｐｈＧＭ９１的ＤＨ５α菌在４２℃诱导，表达了预期的１５ＫＤ的蛋白，约占菌体总蛋白的２０％，并探讨了不同菌株对表达水平的影响。用ＭＴＴ法测定所表达的ＧＭ-ＣＳＦ生物活性，比活性可达到１×１０~７ＩＵ／ｍｇ，表明所表达的ＧＭ-ＣＳＦ具有生物学作用。     

人脑源性神经营养因子及神经营养素—3重组腺病毒的构建及鉴定

目的：构建人脑源性神经营养因子和神经营养素－３重组腺病毒，使其携带神经营养因子感染神经系统细胞，在局部释放有生物活性的营养因子。方法：Ｕ针人全长ＢＤＮＦ与ＮＴ３基因分别插入ｐＣＤＮＡ３载体，然后将表达序列ＣＭＶ－ＢＤＮＦ－ＢＧＨｐＡ和ＣＭＶ－ＮＴ３－ＢＧＨｐＡ切下，插入Ｅ１区缺失的腺病毒载体ｐＨΔＥｌｓｐ１Ａ中，与ｐＪＭ１７共转染２９３细胞，通过同源重组产生重组腺病毒。结果和结论：经过二次ＣｓＣｌ     

人白细胞介素15基因的分离及其在大肠杆菌中的克隆与表达

目的：分离编码人ｈＩＬ－１５的ｃＤＮＡ并大肠杆菌中克隆与表达，方法：采用ＰＨＡ刺激人外周血白细胞提取ｍＲＮＡ，并利用ＲＴ－ＰＣＲ方法获得ｈＩＬ－１５的ｃＤＮＡ将其定向插入ＰＵＣ１９载体中，ＤＮＡ序列分析表明分离的ｈＩＬ－１５的序列与文献报道一致，以ｐＢＶ２２０为表达载体构建并筛选出重组于ｐＢＶｈＩＬ－１５；重组子转化Ｅ．ｃｏｌｉＤＨ５α菌株，经４２℃诱导表达。结果：相对分子质量为１４０００的ｈＩＬ     

我国登革2型病毒株NS1基因的克隆与表达

目的：通过对登革病毒ＮＳ１基因的表达,研究表达的ＮＳ１蛋白的抗原性,为研制新型登革疫苗提供依据。方法：采用ＲＴ－ＰＣＲ和分子克隆技术将扩增的ＮＳ１基因插入到ｐＢＶ２２０载体中,通过温控诱导进行外源蛋白的表达,用ＳＤＳ－ＰＡＧＥ和蛋白质印迹法分别测定表达产物的相对分子质量及特异性。结果：获得正向插入的ＮＳ１－ｐＢＶ２２０重组质普,表达产物的相对分子质量约为５１０００,与预期大小一致,并且可与登革２型     

rhTPO的克隆及其在原核细胞中的表达

为了更加简便有效地得到重组人血小板生长因子（ｒｈＴＰＯ），以治疗放射及化学药物等所致骨髓损伤造成的血小板减少与缺乏，缓解临床上的出血症状，降低死亡率。方法本实验从６月龄人胚胎肝脏细胞中分离ｍＲＮＡ，设计合成特异性引物，经逆转录，套式ＰＣＲ等步骤克隆出了人ＴＰＯ全基因。将人ＴＰＯ基因亚克隆至原核细胞表达载体ｐＢＶ２２０，形成了重组表达质粒ｐＢＶ２２０／ＴＰＯ，以该重组质粒转化了原核细胞大肠杆菌ＤＨ５α，应用４２℃热诱导表达目的蛋白。结果经ＳＤＳ－聚丙烯酰胺凝胶电泳检测，得到了ＴＰＯ在原核细胞中的表达，其分子量与目的一致。结论ｈＴＰＯ可以在原核细胞得到良好的表达，为临床血小板减少症的生物治疗奠定了基础。     

人睫状神经营养因子的原核表达及其初步纯化

目的：优化条件实现人睫状神经营养因子（ｈｕｍａｎｃｉｌｉａｒｙｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ,ｈＣＮＴＦ）ｃＤＮＡ在大肠杆菌中的高效表达。方法：ＣＮＴＦ克隆进ｐＢＶ２２０后,表达,凝胶过滤纯化,１０日龄鸡背根神经节（Ｅ１０ＤＲＧｓ）测定其生物活性。结果：ＣＮＴＦ在ＨＢ１０１中高效表达,并具有生物活性。结论：ＳＤＳ－ＰＡＧＥ观察到相对分子质量约为２４×１０３的预期表达产物,优化表达条件后发现其在ＨＢ１０１中的表达状况最好,用凝胶过滤纯化的重组蛋白能促进Ｅ１０ＤＲＧｓ的生长     

我国登革2型病毒prM-E基因与SFV病毒载体重组RNA的构建

目的：构建我国登革２型病毒４３株ｐｒＭ－Ｅ基因的甲病毒（Ｓｅｍｌｉｋｉ ｆｏｒｅｓｔ ｖｉｒｕｓ,ＳＦＶ）重组ＲＮＡ,为登革新型疫苗的研究奠定基础。方法：首先将含有多种稀有限制酶位点的接头插入ｐＳＦＶ载体的多克隆位点,再把ｐｒＭ－Ｅ基因克隆至该载体中。将获得的重组质粒ＤＮＡ经体外转录后,通过电穿孔法将其转录的ＲＮＡ产物导入宿主细胞,并采用间接免疫荧光法检测ｐｒＭ－Ｅ基因的表达。结果：已获得含多种酶位点的ｐＳＦＶ病毒载体,并且所构建的含ｐｒＭ－Ｅ基因的重组ｐＳＦＶ病毒ＲＮＡ,在宿主细胞中的表达产物可与登革２型病毒特异抗体起反应。结论：构建的含ｐｒＭ－Ｅ基因的重组ｐＳＦＶ病毒ＲＮＡ可表达我国登革２型病毒株的特异蛋白。     

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）