重组血管内皮细胞生长因子在大肠杆菌的高效表达

目的:使重组血管内皮细胞生长因子(VEGF)在大肠杆菌中得到高效表达.方法:通过构建表达重组VEGF的质粒PRL621/VEGF,并在大肠杆菌中高效表达.结果:表达量约占菌体总蛋白的40%.对形成包含体的表达产物进行变性,初步复性处理,得到重组人VEGF粗提液,鸡胚绒毛尿囊膜试验表明有促血管生长活性,N-端15个氨基酸序列分析结果,与天然VEGF蛋白质相应序列一致.     

地高辛素标记的DNA探针快速鉴定产不耐热肠毒素大肠杆菌的研究

用地高辛素标记的６７０ｂＰ－ＬＴ－ＤＮＡ片段作为探针，用菌落原位杂交法对９株标准参考株进行检测，结果全部相符。对７０株从临床分离的菌株也进行菌落原位杂交．并与３２－Ｐ标记的探针进行比较．结果地高辛素探针的特异性为９７．７％，敏感性为１００％，符合率为９８．６％。从含正常肠道菌的粪便中可以直接检出ＬＴ－ＥＴＥＣ而无须经纯培养。定量试验表明本法最低检出量为６ＣｆｕＬＴ－ＥＴＥＣ／斑点。整个检测过程短．仅须２４～３０ｈ。探针的稳定性好，有效期可长达１年。     

O-GlcNAc糖基转移酶在原核和真核细胞中的表达和提纯

目的 :本文为进一步研究蛋白 O- Glc NAc糖基化修饰提供重要资源。方法 :为了探索制备纯化具有生物学活性的重组 OGT的有效方法 ,本文用大肠杆菌和 Hi5昆虫细胞表达并纯化具有活性的重组 OGT。携带人 OGTc DNA的 PET32 a质粒在大肠杆菌中表达出带有 1个 S- Tag的重组人 OGT融合蛋白 ,然后用与 S-蛋白质偶联的琼脂糖凝胶颗粒从细菌裂解液中亲和纯化 OGT融合蛋白。结果 :其纯化的 OGT在电泳中显现单一条带并具有生物活性 ,但其活性在洗脱后丧失。在 Hi5昆虫细胞中表达的鼠肝 OGT带有一 His6 tag,经镍离子螯合柱纯化后 ,其比活性达到 0 .88nmol· min- 1 · mg- 1 OGT。结论 :在这两个表达系统中制备重组 OGT各有利弊。     

自杀基因联合放射对食管癌细胞株的实验研究

目的自杀基因具有能将无细胞毒性的前体药物转化为有细胞毒性作用的药物而使细胞死亡,通过观察大肠杆菌胞嘧啶脱氨酶/氟胞嘧啶(CD/5-FC)自杀基因系统对食管癌EC109细胞杀伤效应及与放射联合应用对食管癌肿瘤细胞协同杀伤效应。方法采用PCR方法从大肠杆菌基因组DNA中扩增出CD基因;构建重组真核载体pcDNA3.1-CD;用脂质体转染法转染EC109细胞;观察CD/5-FC体系对EC109细胞的杀伤效应和旁观者效应及对放射的增敏效应。结果RT-PCR分析结果表明CD基因已转入EC109细胞并表达。离体实验证实5-FC对转CD基因后的食管癌细胞株有明显的细胞毒作用,含5%的转基因食管癌细胞加入5-FC后的细胞存活比率为41.8%±14.2%,低于对照组的94.6%±4.3%结果(t=3.14,P<0.05);加入10%的转基因细胞的存活比率为37.8%±4.4%,低于对照组的95.6%±5.4%结果(t=9.75,P<0.01)。CD/5-FC体系对肿瘤细胞放射增敏效果明显,加前药5-FC及放射组细胞存活曲线低于单纯加前药5-FC对照组或单纯放射对照组,重复测量数据方差分析结果显示F=11.50,P<0.01;治疗组分别与5-FC对照组及单纯放射对照组相比F值分别为4.11、10.53,P值分别<0.05、0.01。结论CD/5-FC体系的旁观者效应和放射增敏效果明显。     

Tubo-ovarian Abscess in Virginal Adolescent Females: A Case Report and Review of the Literature

BackgroundA tubo-ovarian abscess (TOA) is a serious complication of pelvic inflammatory disease (PID), predominantly polymicrobial and present in sexually active women. TOA in virginal adolescent females are extremely rare but have serious and lifelong consequences.CaseA 13 y.o. virginal female presented to the Emergency Room of a tertiary care pediatric hospital with abdominal pain and vomiting. Imaging suggested bowel compromise with potential perforation. An exploratory laparotomy revealed TOA which grew Escherichia Coli. This is the first reported case of Escherichia Coli TOA due to suspected bowel translocation.ConclusionReview of the literature identified 8 cases of TOA in virginal adolescents. Given the severity of outcomes following TOA, this pathology should be considered in the differential diagnosis of virginal adolescents who present with fever and abdominal pain. If suspected, a prompt gynecology consult should be initiated, followed by a first line antibiotic therapy and when indicated, surgical drainage.     

Adult Hemolytic-Uremic Syndrome Associated with Urosepsis Due to Shigatoxin-Producing Escherichia Coli O138:H-

We report the case of a 62-year-old man without prodromal symptoms who developed a hemolytic-uremic syndrome (HUS) one week after the diagnosis of an acute bacteremic urinary infection (UTI). In this patient, the E. coli isolated in blood cultures was a non-O157:H7 Shigatoxin-producing strain that could subsequently be identified as O138:H-. This is a strain that is normally found in pigs and that has never been isolated in humans previously. UTI-related HUS is a rare event, as until now, only 14 pediatric and 3 adult cases have been reported. Indeed, this new case, besides its interesting microbiological aspects, should heighten our awareness of UTI-related HUS as a rare but real condition, not only in young children but also in adult patients. This should emphasize the necessity to search actively for other sources of Shigatoxin-producing E. coli in patients presenting with HUS without gastrointestinal symptoms.     

BONE-INDUCTIVE EFFICACY OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2 EXPRESSED IN ESCHERICHIA COLI: AN EXPERIMENTAL STUDY IN RAT MANDIBULAR DEFECTS

Because to our knowledge the efficacy of prokaryotically expressed recombinant human bone morphogenetic proteins (rhBMP) to promote orthotopic osteogenesis has not previously been investigated, our aim was to test the efficacy of rhBMP-2 produced in Escherichia coli to promote bone healing in a standardised experimental bone healing model in rat mandibles. Different doses of rhBMP-2 were delivered in an absorbable collagen sponge carrier, and microporous barrier membranes were placed over half the number of defects in each treatment group, thereby making intraosseous cells the only recruitment source for new osteogenic cells. Results were evaluated by computerised image analysis after 12 and 24 days. The relative efficacy of rhBMP-2 preparations of different purity was also compared. E coli-produced rhBMP-2 stimulated bone healing, but its efficacy was estimated to be about one order of magnitude less than that of rhBMP-2 expressed in eukaryotic cells. We conclude that bacterially expressed rhBMP-2 is osteogenic in vivo, although higher doses will be required than of rhBMP-2 expressed in mammalian cell lines.     

产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌的检测与耐药性分析

目的:了解大肠埃希菌和肺炎克雷伯菌产ESBLs的检出率及耐药性。方法:采用2007年CLSI/NCCLS推荐的初筛试验方法,从352株大肠埃希菌和肺炎克雷伯菌中筛选出可疑产ESBLs细菌,并用验证试验加以确证,药敏试验采用Kirby-Bauer法。结果:大肠埃希菌和肺炎克雷伯菌ESBLs的检出率分别为42.68%(105/246)和52.83%(56/106);产ESBLs株对亚胺培南高度敏感,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦和头孢西丁耐药率较低,对其他抗菌药物的耐药率均较不产ESBLs株高。结论:重视产ESBLs细菌的检测,根椐药敏试验合理用药,亚胺培南、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦和头孢西丁是目前治疗ESBLs株的有效药物。     

加强对口岸供机食品监管有效性的探讨

目的通过该案例流行病学调查分析,加强对供机食品监管的有效性。方法对温州航空食品实业公司的供机食品:正餐冷荤、正餐水果和点心面包,进行卫生监督检验。结果两份供机食品样品检测大肠菌群严重超过卫生标准。结论检验检疫机关应当加强对航空供机食品卫生监督,改善食品加工场所,作业流程,提高员工个人卫生意识,防止病源微生物污染供机食品,保证食品卫生安全。