人层黏连蛋白α4链LG1-3组件的克隆及酵母表达

目的:利用巴斯德毕赤酵母表达系统获得重组的人层黏连蛋白LG1-3组件蛋白,为进一步研究LG1-3的结构和功能间的关系奠定基础。方法:利用RT-PCR从人胎盘组织总RNA中扩增LG1-3组件cDNA,并重组入pPICZαA,电转化入毕赤酵母GS115,利用甲醇诱导表达目的蛋白。结果和结论:获得了1 800 bp LG1-3组件cDNA,利用体外定点突变将SacⅠ酶切位点GAGCTC突变为GAGCGC,经SDS-PAGE和W estern印迹检测到相对分子质量为67.48×103的LG1-3组件蛋白的表达。     

一种快速鉴定重组酵母克隆的方法

甲基营养型毕赤酵母巴斯德毕赤酵母(Ｐｉｃｈｉａｐａｓｔｏｒｉｓ)是目前较为有效的外源基因表达系统,可实现目的蛋白的高效分泌表达,且可对目的蛋白进行糖基化加工等[1]。毕赤酵母表达系统利用的是乙醇氧化酶基因(Ａｏｘ1)启动子作为强诱导型启动子,可被甲醇诱导。宿主菌ＧＳ115在Ｈｉｓ4基因位点被突变,因而只有当携有外源基因和Ｈｉｓ4基因的表达载体(如ｐＰＩＣ9或ｐＨＩＬＳ1)与酵母染色体在Ａｏｘ1基因处发生同源重组后,酵母克隆才能在组氨酸缺陷培养基(ＭＤ)平板上生长。而此时由于酵母基因组上Ａｏｘ1基因被取代,重组克隆仅能利用…     

血小板糖蛋白GPⅠbαvWF结合域在酵母中的表达鉴定

目的：实现重组糖蛋白GPⅠbα vWF结合域在巴氏毕赤酵母中的表达。方法：PCR从重组质粒pcDNA3．1-GPⅠbα中扩增GPⅠbα vWF结合域基因,以pPIC9k为表达载体,构建包含该目的基因的酵母重组质粒,转染毕赤酵母GS115,以G418筛选抗性克隆,甲醇诱导表达,SDS-PAGE与点印迹鉴定表达产物。结果：获得编码正确的重组质粒pPIC9k-GF302,电转化GS115,以G418筛选到阳性克隆,诱导表达培养上清进行点印迹,结果表明表达蛋白能够与抗GPⅠbα单克隆抗体结合。结论：获得了表达重组蛋白质GPⅠbα vWF结合域的巴氏毕赤酵母菌株以及重组蛋白质GP302。     

利用基因敲除技术缺失毕赤酵母羧肽酶Y基因的初步研究

目的通过基因敲除技术缺失毕赤酵母编码的羧肽酶Y（carboxypeptidase Y,CPY）基因cpy,观察其对毕赤酵母生长和外源蛋白表达的影响。方法通过PCR方法扩增出内部含有潮霉素B抗性基因的cpy同源序列;通过电转法将同源序列转入GS115-EH感受态细胞中,并用潮霉素B筛选出阳性克隆。用平板显色法测定阳性克隆菌的羧肽酶活性,同时用PCR法鉴定阳性克隆菌的cpy基因是否缺失。对cpy基因缺失的GS115-EH△cpy菌,分别用YPD和BMGY-BMMY培养研究其生长特性;用BMMY培养基进行甲醇诱导表达外源蛋白,观察其蛋白表达的特性。结果 GS115-EH△cpy的羧肽酶基因cpy被成功敲除,羧肽酶活性丧失。YPD培养条件下,GS115-EH△cpy生长后期的D600明显低于GS115-EH,而用BMMY诱导培养基,GS115-EH△cpy的生长明显改善,但仍然低于对照菌。蛋白电泳结果显示甲醇诱导GS115-EH△cpy的蛋白表达量也低于对照菌。结论 GS115-EH△cpy的cpy基因被成功敲除,在YPD培养条件下,其生长低于对照菌,在BMMY培养条件下,其生长明显改善,但仍然较对照菌差,而且其外源蛋白的表达也低于对照菌。     

人ω干扰素在巴斯德毕赤酵母中的表达及纯化

目的 :在巴斯德毕赤酵母中表达及纯化人ω干扰素 (interferonω ,IFN_ω)以对其功能进行深入研究。方法 :用PCR方法扩增人IFN_ω基因 ,与分泌型酵母表达载体pMEX9K重组 ,经酶切、PCR和序列测定证实重组表达载体pMEX9Kω构建正确。将重组载体pMEX9Kω用SalⅠ酶切后 ,转化毕赤酵母GS115。得到的转化子经诱导表达后在发酵上清中有人IFN_ω的表达 ,表达量为 4× 10 9U L。经过ButylSepharose 4FF疏水层析柱 ,SPSepharoseFF离子交换柱和Superdex 75凝胶过滤三步层析纯化 ,利用反相HPLC分析纯化的重组蛋白。结果 :得到了纯度 >99%的重组IFN_ω。N端氨基酸序列测定表明重组人IFN_ω具有正确的N端氨基酸残基顺序 ,相对分子质量为 2 2 0 0 0 ,并具有同天然蛋白相似的比活性。结论 :在毕赤酵母中成功地表达并纯化得到了同天然蛋白具有相似的比活性的人IFN_ω。     

长链胰岛素样生长因子1在毕赤酵母中的表达、纯化及活性分析

目的:在毕赤酵母KM71中分泌表达长链-Arg3-胰岛素样生长因子1(LR3IGF-1),并探讨其体外生物活性.方法:人工合成LR3IGF-1基因,构建表达载体pCαA-LR3IGF-1,转化毕赤酵母KM71.筛选重组工程菌株,经诱导表达并从上清中纯化目的蛋白,鉴定其生物活性.结果:诱导上清中存在一8×103 左右的条带,经初步鉴定为目的蛋白条带.每升培养基经纯化后能得到40 mg目的蛋白.纯化后的目的蛋白具有明显的体外刺激NIH 3T3细胞增殖的作用.结论:建立了LR3IGF-1在毕赤酵母 KM71中表达及纯化方法,该方法能获得具有生物学活性的高纯度LR3IGF-1,为LR3IGF-1的后续研究及临床应用奠定了基础.     

κ阿片受体激动剂U45088 H对大鼠体外循环后认知功能及PKA-CREB信号通路影响

目的探讨κ阿片受体(KORs)激动剂U45088H对大鼠体外循环(CPB)后认知功能及PKA-CREB信号通路的影响。方法选取成年雄性SD大鼠40只为研究对象。采用随机数字表法分为假手术(S)组、CPB(C)组、CPB+U50488H(K)组、CPB+KORs拮抗剂Nor-BNI+U50488H(N)组与CPB+PKA拮抗剂H89+U50488H(H)组,每组各8只。S组不建模型,其余4组各CPB 60 min。K组CPB前30 min静脉注射U50488H 1.5 mg/kg;N组CPB前1 h静脉注射Nor-BNI 2 mg/kg,CPB前30 min静脉注射U50488 1.5 mg/kg;H组CPB前1 h侧脑室注射H89 5μl,CPB前30 min静脉注射U50488H 1.5 mg/kg。1 d后水迷宫法检测大鼠认知功能,TUNEL法观察海马组织细胞凋亡,Western-blot法检测海马组织p-PKA/PKA、p-CREB/CREB蛋白表达情况。结果与S组比较,C、K、N及H组大鼠水迷宫逃避潜伏期均延长,穿越平台次数、游泳距离及停留时间均减少,差异有统计学意义(P<0.05)。与S组比较,C、K、N及H组大鼠海马细胞凋亡率升高,差异有统计学意义(P<0.05)。与S组比较,C、K、N及H组大鼠海马p-PKA/PKA、p-CREB/CREB表达均降低,差异有统计学意义(P<0.05)。结论 KORs可减少大鼠CPB后认知功能障碍的发生,其机制可能与激活PKA-CREB信号通路有关。     

A型肉毒毒素保护性抗原在酵母细胞中的表达与抗原性分析

目的：在酵母系统中实现A型肉毒毒素(BoNTa)Hc基因的可溶性分泌表达。方法：将Hc基因克隆入表达载体pPIC9K，用SacⅠ将重组质粒线性化，电转化巴氏毕赤酵母(Pichia pastoris)GS115细胞，筛选G418抗性阳性整合子，进行Mut^ 表型株甲醇快速利用诱导。结果：经过体外高拷贝整合子的筛选和诱导表达条件的优化，毒素Hc在pH6．5培养基BMMY中实现稳定的分泌表达，最终筛选到蛋白表达量占上清蛋白10％的分泌高表达株。免疫印迹和酶联免疫检测结果显示，重组BoNTa Hc可以识别特异抗肉毒马血清并具良好的特异结合活性，可以诱导小鼠产生特异性抗体。结论：酵母系统来源的具有良好免疫原性的重组BoNTa保护性抗原可以作为有效的候选重组疫苗分子。     

耐辐射奇球菌PprI蛋白质在毕赤酵母菌中的高效表达及纯化

目的 利用真核毕赤酵母高效表达原核的耐辐射奇球菌pprI基因,建立高效表达及纯化PprI蛋白质的技术路线。方法 根据毕赤酵母密码子的偏爱性,改造耐辐射奇球菌pprI基因的编码序列,利用PCR技术全合成改造过的pprI基因,并在其N末端添加一个6×His标签。PCR产物经纯化后克隆到毕赤酵母表达载体pHBM-905A中,利用Cop I和Not I双酶切并回收线性化的目的片段后,转化毕赤酵母GS115菌株。将获得的毕赤酵母转化子诱导表达,SDS-PAGE、Western blot和质谱检测培养上清液,用Ni-NTA柱纯化目的蛋白,BCA法测定蛋白浓度。结果 新合成的耐辐射奇球菌pprI基因编码序列正确,毕赤酵母转化菌株的培养上清液经SDS-PAGE和Western blot均可检测到相对分子质量为43000的目的蛋白条带,经质谱检测证实该目的蛋白为耐辐射奇球菌PprI蛋白。选用Ni-NTA柱获得了大量纯化的PprI蛋白,应用浓度为250 mmol/L的咪唑淋洗时,PprI蛋白洗脱率最高。BCA法测得纯化蛋白浓度为0.35 mg/ml。结论 建立了一种新的适于毕赤酵母表达的耐辐射奇球菌pprI基因,成功构建了分泌表达PprI蛋白质的重组毕赤酵母工程菌株,建立了高效表达目的蛋白的技术路线,并获得了高效表达和纯化的PprI蛋白。     

唾液HRP5第17位密码子反义突变对其在毕赤酵母中表达的影响

目的 在毕赤酵母中表达唾液富组蛋白5(HRP5)基因原始序列hrp5及突变序列hrp5'(Lys17→Asn),比较表达量及抗白色念珠菌活性.方法 选用毕赤酵母偏爱密码子,设计3'端互补、5'端带酶切位点的两对引物,PCR合成hrp5和hrp5',克隆至pPICZα-A,转化大肠杆菌,将酶切、测序鉴定的阳性重组质粒pPICZα-A-hrp5和pPICZα-A-hrp5'经Sac I线性化后电击转化GS115,筛选阳性菌落,PCR鉴定,甲醇诱导表达,比较抗菌活性,测定表达量.结果 hrp5及hrp 5'正确整合到GS115基因组中,突变前后表达产物与HRP5标准品活性相当,表达量分别为4μmol/L、5μmol/L.结论 HRP5在毕赤酵母中成功表达,活性区Lys17突变为Asn使表达量提高25%,而对其杀菌活性无影响.     

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.     

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.     

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.     

The role of interventional radiology in trauma

Introduction Interventional Radiology has evolved into a specialty having enormous input into the care of the traumatized patient.In all hospitals,regardless of size,the Interventional Radiologist must consider their relationships with the trauma service in order to     

人体中的镭-226、镭-228、钋-210、铅-210

本文报道了广东阳江高本底地区6名、对照地区8名人尸体的骨²²⁶Ra、²²⁶Ra的浓度以及部分居民内脏器官中。²¹⁰Po、²¹⁰Pb的浓度。结果轰明阳江高本底地区和对照地区居民骨镭-226、镭-228的浓度分别为29.9pCi/kg, 26.9pCi/kgl 8.7pCi/kg, 8.2pCi/kg.由此估算出阳江高本底地区屠民骨中²²⁶Ra、²²⁸Ra的负薄璧及对骨衬、骨髓所产生的剂量当量分别为对照地区民民的3.4倍, 3.3倍。两地区居民内脏器官中^{210Po、210Pb的测定分析铡数较少但仍看出, 高本底地区均明显高于对照地区.}     

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).