人β-防御素3在大肠杆菌中的融合表达及其抗微生物活性的初步分析

目的：在大肠杆菌系统中表达有活性的人β-防御素3(hBD3)。方法：从pGE-T-hBD3重组质粒PCR获得hBD3成熟肽编码区基因，插入pGEX-4T-1构建pGEX-4T-1-hBD3融合表达载体，转化E．coli DH5α宿主菌，进行IPTG诱导，诱导菌经超声裂解后获得包涵体，包涵体经溶解、变性、复性和纯化处理后获得GST-hBD3融合蛋白，再经凝血酶切割。获得纯化的重组hBD3(rhBD3)。对rhBD3采用最小抑菌浓度测定法测定其对金黄色葡萄球菌和大肠杆菌的抗菌活性。结果：以与GST融合表达的方式，运用pGEX-4T-1系统在大肠杆菌中表达了hBD3，融合蛋白以包涵体形式存在。融合蛋白经凝血酶处理使删释放出来，其对金黄色葡萄球菌的MIC为4μg/ml；对大肠杆菌的MIC为8μg／ml。结论：采用pGEX-4T-1融合表达系统在大肠杆菌中成功地表达了有活性的rhBD3。     

新型重组IL-6 D24-PE40KDEL外毒素融合蛋白复性及纯化

目的：对高效表达的重组IL-6 D24-PE40KDEL外毒素融合蛋白进行包涵体的分离、变性与复性以及纯化。方法：对表达菌体进行超声破碎、洗涤、氧化还原法变性及复性，经离子交换层析。结果：工程菌HB101／pE01T的表达产物是以包涵体形式存在，菌体的破碎以高渗蔗糖溶液进行预处理后，再超声破碎效果最好；包涵体的洗涤则先用Triton-X-100洗2次，再用8mol／L尿素洗涤一次效果更优；最佳变性条件为在7mol／L盐酸胍中加入0．065mmol／L的DTT和0．2mol／L的盐酸精氨酸；最佳复性奈件为在10℃复性保持24h，蛋白浓度保持在30～80μg/ml，并需加入0．2mol／L盐酸精氨酸；利用谷胱甘肽的氧化还原法，当氧化型谷胱甘肽与还原型谷胱甘肽的浓度比在2：1时所获得的活性成分得率最高；利用强弱离子交换结合的方法能获得纯度为95％的目的蛋白，所得融合蛋白可与IL-6及PEA抗体相结合并能特异性地杀伤高表达IL-6R的人多发性骨髓瘤细胞。结论：本研究获得重组IL-6D24-PE40KDEL外毒素融合蛋白包涵体的分离、变性与复性条件和一条能获得大量高纯度该融合蛋白的纯化路线。     

小鼠及人源化鼠抗人交联纤维蛋白单链抗体的表达与纯化

目的：表达并纯化小鼠及人源化鼠抗人交联纤维蛋白单链抗体,并初步分析比较两者的体外生物活性。方法;经ＤＮＡ重组的构建重组表达质粒,经ＩＰＴＧ诱导,在大肠杆菌中高表达两种单链抗体,用８ｍｏｌ／Ｌ尿素溶解包涵体后经ＩＭＡＣ纯化表达产物,并用凝血酶切除Ｎ端融合的（Ｈｉｓ）６,纯化的表达产物经复性后ＥＬＩＳＡ检测其活性,结果：两种单链抗体在大肠杆菌中表达量占全菌蛋白的５０％以上,经变性,纯化后纯度可达９７％     

牛蛙核糖核酸酶对体外培养的人肝癌细胞的杀伤作用

目的：利用大肠杆菌系统表达牛蛙核糖核酸酶（RC—RNase），观察其对人肝癌细胞的体外杀伤作用。方法：构建PE-q32-RC—RNase重组原核表达载体，转化人大肠杆菌BL21（DE3）plyS中并利用IPTG诱导表达。工程菌经超声碎菌、离心后，表达产物在变性条件下经Ni—NTA—agzrose亲合层析法纯化及复性，细胞毒试验检测其对体外培养的人肝癌细胞的杀伤活性。结果：经IPTG诱导后，大肠杆菌中表达的RC—RNase融合蛋白相对分子量（Mr）约为31KD。与预期大小基本相一致，主要以包涵体的形式存在。经纯化及复性后，获得纯度达到95％以上且对体外培养的人肝癌细胞具有明确杀伤活性的RC—RNase融合蛋白。结论：在大肠杆菌中表达的RC—RNase融合蛋白对体外培养的肝癌细胞具有一定的杀伤作用。     

RAP结合肽-hFcγ1融合蛋白的表达与初步活性鉴定

目的：将肽库筛选到的正调控金黄色葡萄球菌(金葡菌)外毒素分泌的关键分子RAP(RNA Ⅲ activating protein)的结合肽与人IgG1抗体Fc片段(hFcγ1)融合表达，检测其干扰金葡茵外分泌毒素产生的活性。方法：根据噬茵体肽库筛选到的RAP结合肽序列，选用大肠杆菌偏爱的密码子合成基因片段，将其融合在人IgGl抗体Fc片段基因序列的5’端，构建pET-rapbp-fc融合表达载体，转化大肠杆菌并诱导表达。以包涵体形式存在的目的蛋白经变性、复性后。ELISA实验分别证实其与抗人IgG抗体及纯化RAP的结合活性。以MDBK细胞株为模型，MTI实验观察融合蛋白对金葡菌196菌株外分泌毒素水平的影响。结果与结论：融合基因在大肠杆菌中获得表达。复性后，融合蛋白的Fc片段及结合肽部分分别能够与抗人IgG抗体及RAP结合，且能在一定程度上降低金葡菌196株上清中的毒素水平。     

用pProEXHT载体进行人FLT3配体基因的表达与产物的亲和层析纯化

目的 :探讨寡聚组氨酸多肽与目标蛋白融合基因的表达及其产物的金属离子螯合亲和层析纯化方法。方法 :将人FLT3配体 (FL)胞外段cDNA连入 pProEXHT载体 ,转入大肠杆菌实现表达。分离包涵体并进行复性处理后 ,通过Ni2 离子螯合亲和层析纯化融合蛋白表达产物。结果和结论 :6×组氨酸 FL融合蛋白的表达量约占菌体蛋白总量的 15% ,用Ni2 离子螯合亲和层析纯化表达产物 ,一次过柱的纯度可达 90 %以上 ,操作程序简便省时 ,是日后FL基因工程产品大规模制备的可行途径     

NK细胞免疫球蛋白样受体KIR3DL1胞外区的表达及纯化

目的 表达并纯化NK细胞免疫球蛋白样受体KIR3DL1胞外区.方法 以pUC57-KIR3DL1为模板,PCR扩增KIR3DL1胞外区序列,与pGEM-T载体进行A-T克隆;测序正确后,采用定向克隆的方法将KIR3DL11胞外区基因连接到pET28a-DsbA载体上,成功构建了pET28a-DsbA/KIR3DL1重组质粒.将重组质粒导入大肠杆菌BL21(DE3),IPTG诱导DsbA-KIR3DL1融合蛋白表达,溶解于8mol/L尿素中的包涵体经Ni-NTA琼脂糖亲和层析纯化,成功进行复性,再经Superdex 75凝胶层析柱进一步纯化,SDS-PAGE和Western blot鉴定目的 蛋白的表达及纯化.结果 表达产物呈部分可溶性表达,包涵体纯化后证实获得纯度95%以上的DsbA-KIR3DS1融合蛋白.结论 KIR3DL1胞外区的成功表达及纯化为进一步研究KIR3DL1与相应配体之间的相互作用奠定了基础.     

新型重组人源抗狂犬病毒糖蛋白单链抗体的制备及活性分析

目的：利用大肠杆菌表达新型人源抗狂犬病毒糖蛋白单链抗体ScFv,并验证其活性。方法：采用基因融合获得ScFv基因,构建重组表达质粒pET-22b（＋）-ScFv,转化大肠杆菌BL21（DE3）,经IPTG诱导获得高效表达。结果：Western印迹显示目的蛋白表达正确,表达产物以包涵体形式存在,经Ni-NTA柱纯化和体外复性,获得纯度达90%的ScFv蛋白。ELISA结果显示在PBS及人血清中ScFv的结合稳定性有所提高,流式细胞术证明目的蛋白能靶向结合狂犬病毒,通过中和效价测定实验测得ScFv的中和效价为40 U/mg。结论：成功利用原核表达系统实现了对人源抗GPRV ScFv的表达,并且具有一定的中和活性。     

人斯钙素1在大肠杆菌中的重组表达与活性鉴定

目的：通过原核表达的方法得到有活性的斯钙素1（ STC1）重组蛋白。方法将优化合成的STC1 DNA片段克隆到表达载体pET-32 b（＋）上,重组质粒转入大肠杆菌诱导表达,在变性条件下纯化包涵体,经透析复性得到可溶的斯钙素1融合蛋白,凝血酶酶切后用高亲和镍离子树脂吸附多余多肽获得目的蛋白。 Western印迹分析验证目的蛋白免疫活性。动物实验检测目的蛋白的生物学活性。结果构建了pET-32b（＋）-STC1表达载体,表达并纯化了STC1融合蛋白包涵体,经复性、凝血酶切和纯化后获得可溶的目的蛋白。 Western印迹分析验证正确。生物学活性检测表明重组STC1能增加大鼠排泄系统对磷酸盐的重吸收。结论获得了具有生物学活性的斯钙素1重组蛋白,为制备STC1单克隆抗体和进一步研究STC1与肿瘤治疗的关系奠定了基础。     

青霉素结合蛋白2a C-末端在大肠杆菌M15中的表达、纯化及鉴定

目的构建含目的基因mecA C末端的重组子并在大肠杆菌M15中进行蛋白表达。方法采用PCR方法扩增mecA C末端DNA，将其与表达载体pQE30连接；再通过PCR及双酶切鉴定连接产物。将含目的基因片段的阳性重组质粒转化大肠杆菌M15后，用1mmol／L的IPTG进行诱导表达。应用Ni-NTA琼脂糖进行亲和层析纯化目的蛋白，并用SDS-PAGE和Western blotting对表达产物进行鉴定。结果成功构建pQE30-mecA重组表达质粒，转化大肠杆菌M15，经IPTG诱导表达，亲和层析纯化获得分子量为39．7kDa的蛋白；Western blotting和肽指纹图谱证实该蛋白为带有多聚组氨酸标签的融合蛋白。结论在大肠杆菌内成功表达具有活性的PBP2a，为后期研究和开发抗MRSA药物奠定了基础。     

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.     

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）     

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.