结核分枝杆菌Ag85A蛋白的原核表达、纯化和免疫反应性

目的通过原核表达获得结核分枝杆菌Ag85A蛋白。方法用PCR从结核分枝杆菌H37Rv菌株中扩增出编码Ag85A的fbpA基因,克隆入原核表达载体pProEXHTb,产生重组质粒pPro85A后,转化至大肠杆菌感受态细胞BL21并诱导大量表达。用镍纯化系统纯化重组Ag85A蛋白,用不同分枝杆菌感染的小鼠血清通过ELISA确定其免疫反应性。利用PCR技术鉴定fbpA基因在不同分枝杆菌的分布。结果32 ku的Ag85A蛋白获得高效表达和纯化。表达Ag85A蛋白的fbpA基因在结核分枝杆菌H37Rv、H37Ra、BCG、草分枝杆菌、土地分枝杆菌、耻垢分枝杆菌和次要分枝杆菌中均有表达,但在牝牛分枝杆菌中未表达。结核病患者和结核分枝杆菌毒株H37Rv感染小鼠血清所产生的抗Ag85A抗体滴度最高。结论重组Ag85A蛋白已成功表达纯化,并保留了免疫反应性。     

胶原/PLLA纳米粒子复合海绵的制备及其生物相容性评价

通过紫外和自由基聚合在PLLA上接枝聚丙烯酸（PAA）,制备了平均粒径316nm,zeta电位-39.88mV的表面亲水性PLLA纳米粒子,与胶原溶液混合通过冷冻干燥法制备了胶原/PLLA纳米粒子复合多孔支架。复合支架材料的孔隙率与纯胶原支架相近,而密度、湿态压缩模量有明显提高,降解率降低显著。随着复合支架中PLLA纳米粒子的增加,L929细胞在材料中的播种效率和增殖有明显提高,表明胶原/PLLA纳米粒子复合多孔支架是一种理想的组织工程支架材料。     

结核分枝杆菌融合蛋白和分泌蛋白对人肝癌细胞HepG-2影响和作用

目的探讨结核分枝杆菌融合蛋白对人肝癌细胞HepG-2的抑制作用。方法构建含3种目的基因的表达载体pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6和pProEXHTb-Hsp16.3,分别转入宿主菌E.coli DH5α中,诱导表达后分别获得Ag85B-Hsp16.3、Ag85B-ESAT6和Hsp16.3三种蛋白,纯化后复性。分别作用于肝癌细胞HepG-2,MTT法检测细胞生长情况。结果成功纯化并复性三种蛋白。MTT实验结果显示,三种蛋白均对HepG-2细胞的生长具有抑制作用,抑制强度与蛋白终浓度和作用时间相关,但各蛋白的抑制作用无明显差异。结论结核分枝杆菌的部分分泌蛋白对肝癌细胞HepG-2具有抑制作用。     

结核分枝杆菌Ag85B-Esat6-HspX融合基因真核表达载体的构建

目的构建结核分枝杆菌Ag85B-Esat6-HspX融合基因,并对其在体外真核细胞中进行表达。方法用PCR法从结核分枝杆菌H37Rv株基因组中分别扩增Ag85B、Esat6、HspX基因,插入到pUC19-T载体,序列测定正确后,将融合基因再次克隆到真核表达载体pcDNA3.1(-)。重组质粒经酶切鉴定并测序正确后,用MegaTran1.0转染293T细胞,并用Western-Blot检测目的蛋白的表达。结果 Western-blot检测到分子量大小约65 kDa的目的蛋白。结论成功地构建了结核分枝杆菌Ag85B-Esat6-HspX融合基因的真核表达载体,且该重组载体可在体外真核细胞中获得特异性的表达。     

光致还原制备透明质酸-纳米银复合物

以透明质酸(HA)为分散载体,通过紫外光还原硝酸银制备出了Ag纳米颗粒分散体系。用紫外-可见光分光光度计(UV-Vis)、傅里叶变换红外光谱(FT-IR)、透射电子显微镜(TEM)、动态激光光散射(DLS)及抑菌实验对所得样品的形貌、结构及性质进行表征。结果表明：所制备的Ag纳米颗粒呈球形,平均粒径约为18 nm,在水相中能稳定分散,且对金黄色葡萄球菌(S.aureus)具有明显的抑菌作用。     

Cancer vaccines and carbohydrate epitopes

Tumor-associated carbohydrate antigens (TACA) result from the aberrant glycosylation that is seen with transformation to a tumor cell. The carbohydrate antigens that have been found to be tumor-associated include the mucin related Tn, Sialyl Tn, and Thomsen-Friedenreich antigens, the blood group Lewis related Lewis^Y, Sialyl Lewis^X and Sialyl Lewis^A, and Lewis^X (also known as stage-specific embryonic antigen-1, SSEA-1), the glycosphingolipids Globo H and stage-specific embryonic antigen-3 (SSEA-3), the sialic acid containing glycosphingolipids, the gangliosides GD2, GD3, GM2, fucosyl GM1, and Neu5GcGM3, and polysialic acid. Recent developments have furthered our understanding of the T-independent type II response that is seen in response to carbohydrate antigens. The selection of a vaccine target antigen is based on not only the presence of the antigen in a variety of tumor tissues but also on the role this antigen plays in tumor growth and metastasis. These roles for TACAs are being elucidated. Newly acquired knowledge in understanding the T-independent immune response and in understanding the key roles that carbohydrates play in metastasis are being applied in attempts to develop an effective vaccine response to TACAs. The role of each of the above mentioned carbohydrate antigens in cancer growth and metastasis and vaccine attempts using these antigens will be described.     

电化学Ag量子线在维生素C检测中的应用

目的研究电化学Ag量子线能否高灵敏、高选择性地检测抗维生素C。方法通过自行设计自动控制电路,采用电化学自终止方法制备Ag量子线,原位滴加维生素C溶液后观察通过量子线电流的改变。结果电化学Ag量子线对维生素C有极灵敏的电流响应,其检测限可以达到0.1mmol/L以下,并对作用机理进行了讨论。结论电化学Ag量子线可用于痕量维生素C的检测。     

MEBO与SD - Ag乳膏治疗放射性烧伤临床对比研究

目的 多年来人们一直主张采用磺胺嘧啶银( SD - Ag)乳膏或抗菌药物乳剂治疗放射性烧伤,但该方法疗程偏长,有些患者因久治不愈形成慢性溃疡,本研究旨在比较湿润烧伤膏(MEBO)与磺胺嘧啶银(SD- Ag)乳膏治疗水疱型放射性烧伤的临床疗效,寻找有效的治疗方法.方法 在2006年6月至2010年6月期间,采用随机分组表分组法,对水疱型放射性皮肤烧伤创面分别采用MEBO和SD - Ag乳膏治疗,前者为MEBO组,共计36例(处),后者为SD - Ag组,共计30例(处);主要观察指标为各组创面自行愈合时间和接受植皮手术的创面个数,以及创面所发生的特殊变化.结果 MEBO组(36处)中有34处自行愈合,占总创面个数的94.4％,自愈创面的平均愈合时间为33.4 d±6.5d,另2例经过清创、移植自体皮肤封闭创面,植皮创面个数占本组创面个数的5.6％; SD-Ag组(30处)中有22处自行愈合,自愈创面的平均愈合时间为41.2d±9.9d,另8例(处)经过清创、移植自体皮肤封闭创面,植皮创面个数占本组创面个数的25.7％.结论 MEBO治疗水疱型放射性烧伤的临床疗效优于SD - Ag乳膏,可明显缩短创面愈合时间,降低手术几率.     

Allergens in Hymenoptera venom XIII: Isolation and purification of protein components from three species of vespid venoms

Pure venoms were collected from individual insects of the species Dolichovespula maculata, white-faced hornet, Vespula squamosa, southern yellow jacket, and Polistes exclamans, paper wasp (one species). The venoms were first fractionated by high-resolution gel filtration on a 1.6 m column of Sephadex G-75 superfine, and the components were then purified by high-performance, ion-exchange chromatography on a Mono-S cation exchange column followed by a further gel filtration step. The isolated components were evaluated for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis by use of two different types of silver stains, by assays for enzyme activities, and by immunodiffusion with the use of rabbit antisera. The protein components were isolated in highly purified states by these techniques. Only three significant proteins were found in V. squamous venom: phospholipase (PL) A and B, hyaluronidase (HYAL), and antigen 5 (Ag 5). D. maculata venom contained HYAL, Ag 5, two isozymes of PL A and B, a high-molecular-weight protein, and several trace proteins. No significant amounts of proteases were found in D. maculata venom. P. exclamans venom contained HYAL, PL A and B, Ag 5, a high-molecular-weight protein, and several minor proteins. In all three venoms the PL A and B activities were found to be in the same molecule and did not separate. Trace components with apparent PL A activity were observed in the venoms. The venoms were screened for a variety of esterases, proteases, peptidases, glucosidases, and phosphatases, and none were detected in more than trace amounts. Vespid venoms do not appear to contain significant amounts of acid phosphatases as bee venoms do.