家蚕血淋巴液中重组人粒细胞——巨噬细胞集落刺激因子（rhG—CSF）的制 …

对探索适于昆虫表达体系重组蛋白质的分离、纯化方法，对家蚕细胞表达的重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）的纯化工艺进行了研究。利用离子交换色谱、凝胶过滤色谱及旋转等电聚集电泳等技术，建立了从家蚕血淋巴液中分离、纯化ｒｈＧＭ－ＣＳＦ的工艺路线，经过三步分离，获得了纯度为９７．２４％的目标产品，总回收率达３３．５９％。此法可有效地去除动、植物色素且简单、经济、高效，适于和大，为ｒｈＧＭ     

真鲷肿瘤坏死因子(TNFα)基因的体外重组与纯化研究

将真鲷肿瘤坏死因子(TNFα)基因的成熟肽区域克隆到表达载体pQE30中,并转化到大肠杆菌XL-blue中进行表达。转化子经载体和基因特异性引物进行PCR双向筛选,并将筛选到的阳性克隆经质粒纯化后,进行序列测定。序列测定结果显示,该基因结构正确,且准确插入到表达载体启动子的下游,获得了结构和组成正确的重组子。重、组子经IPTG诱导后,以SDS-PAGE电泳鉴定,电泳结果表明,该基因在大肠杆菌XL-blue中实现了表达。通过对诱导条件的调整,在体外获得了高效表达的重组蛋白,高效表达的重组蛋白约占菌体蛋白的25％-30％。不同诱导时间对重组蛋白影响实验显示,随诱导时间的增加,重组蛋白产量逐渐增高,经4h诱导,其表达量可以达到平台期,过长时间的诱导,对表达产量没有影响。重组蛋白经镍金属亲和层析纯化,获得了纯度较高的重组蛋白。采用交换缓冲液的方法,用Sephadex G150,对重组蛋白进行脱盐复性,使重组蛋白得到了进一步纯化。     

C端带多聚组氨酸标签的重组小鼠基质细胞衍生因子-1α原核系统的表达及分离纯化

在构建SDF-1原核表达系统的基础上，探索对其活性表达产物分离纯化的技术路线。将从小鼠骨髓组织克隆出的SDF-1a成熟肽基因插入原核表达质粒pET101／D-FOFO，用以转化大肠杆菌B1221Star^TM菌株，经SDS-PAGE进行诱导表达。SDS-PAGE和Western Blot检测证实：其表达产物在约11．6kD处有明显条带显示，其大小与预测的重组SDF-1a／His分子量一致。采用Ni^2 -Resin固相亲和层析法对由该系统表达的C端带6Xhis标签的重组SDF-1a／His进行分离纯化，纯度达92％。体外活性检测结果表明：重组小鼠SDF1a／His对T细胞有明显趋化和促生存作用；能有效诱导Jurkat细胞ERK磷酸化。     

CSF－1R部分基因在大肠杆菌中表达、抗融合蛋白血清制备及丝氨酸／苏氨酸磷酸化的初步研究

鼠巨噬细胞集落刺激因子－１受体（ｍＣＳＦ－１Ｒ）部分序列与质粒ｐＧＥＸ－２Ｔ谷胱苷肽转移酶（ＧＳＴ）融合，融合蛋白ＧＳＴ－ＣＤ－Ｐｓｔ（胞浆区），ＧＳＴ－ＣＴｅｒｍ（Ｃ－末端）和ＧＳＴ－ＫＩ（激酶插入区）成功地在大肠杆菌ＪＭ１０９株表达。初步结果指出：（１）ＧＳＴ－融合蛋白在体外激酶分析中可以作为底物；（２）由ＰＫＡ导致的磷酸化可能具有生理学意义；（３）ｍＣＳＦ－１Ｒ被ＣＫＩＩ磷酸化。３２Ｐ标记ＧＳＴ－ＣＤ－Ｐｓｔ的磷酸氨基酸分析证实，ｍＣＳＦ－１Ｒ的胞浆区丝氨酸上被磷酸化，已制备抗ＧＳＴ－ＣＴｅｒｍ，ＧＳＴ－ＫＩ和ＧＳＴ－ＣＤ－Ｐｓｔ兔抗体。抗血清的筛选通过野生型３２Ｄ－ＣＳＦ－１Ｒ转染子免疫沉淀进行。     

真鲷(Pagrosomus major)卵黄原蛋白的分离纯化及抗血清制备

采用分子筛和离子交换两步层析法分离纯化了真鲷(Pagrosomus major)的卵黄原蛋白(VTG),并采用非变性和变性聚丙烯酰胺凝胶电泳测定了真鲷VTG的分子量,发现真鲷VTG在非变性条件下的分子量大约为450kDa;在SDS变性条件下,两条亚基的分子量大约分别为170kDa和120kDa。Western-blot检测结果表明这两条亚基都能够与真鲷VTG多克隆抗血清产生免疫反应,而真鲷VTG的170kDa亚基是检测真鲷VTG的特异性条带,且最低检出浓度为0.1μg L~(-1)。鼠抗真鲷VTG多克隆抗血清对真鲷VTG具有良好的特异性,这一结果为采用真鲷为模式生物检测海洋环境雌激素奠定了实验基础。     

水稻中一种抗真菌蛋白的分离与特性分析

从萌发的水稻种子中分离并纯化了一种能抑制多种病原真菌生长的蛋白（简称为ＲＡＦＰＩ），在马铃薯葡糖琼脂培养基上，２０μｇＲＡＦＰＩ可明显抑制木霉（Ｔｒｉｃｈｏｄｅｒｍａｒｅｅｓｅｉ）菌丝的生长，此蛋白还具有较广的抗真菌菌谱，其分子量为１６．５９ＫＤ，等电点为８．７，组成成分中富含Ｐｒｏ、ＧｌＸ和Ａｓｘ，经测序得到了其Ｎ端２０个氨基酸序列。     

麻疯树核糖体失活蛋白的分离纯化和作用机制研究

从麻疯树种子中分离纯化出一种核糖体失活蛋白，用SDS－PAGE测定其相对分子量为28.2kD,IEF-PAGE计算其等点为8．54左右。体外抑制实验表明，麻疯树核糖体失活蛋白对兔网织红细胞裂解液的蛋白质生物合成有较强的抑制活性。当麻疯树核糖体失活蛋白作用于大鼠肝核糖体经苯胺处理后，在聚丙烯酰胺的尿素变性胶电泳图上能观察到450bp左右的特征性核酸条带，证明麻疯树核糖体失活蛋白是一种RNA N－糖苷酶，从而首次阐明了麻疯树糖体失活蛋白失活核糖体的分子机制。     

The geologic and hydrogeologic setting of the Waste Isolation Pilot Plant

The Waste Isolation Pilot Plant (WIPP) is a mined repository for the permanent disposal of transuranic wastes. It has been constructed by the United States Department of Energy (DOE) in semiarid, sparsely inhabited rangeland in southeastern New Mexico. The disposal area is 655 m below the land surface, in bedded salt of the Late Permian (approximately 255 million-years-old) Salado Formation. The extremely low permeability of the halite and other evaporite rocks provide the primary geologic barrier assuring long-term (10,000-year-plus) isolation of the radioactive waste from the accessible environment. Extensive geologic investigations during site characterization have provided information on the stratigraphy, structure, and natural resources of the region (including hydrocarbons, potash, and groundwater), and have investigated the potential for disruption by processes such as dissolution, salt deformation, tectonic activity, and climate change. Hydrogeologic investigations have documented the physical properties of the evaporite rocks, and have identified the Culebra Dolomite Member of the overlying Rustler Formation as the most transmissive water-bearing unit in the region. If the evaporite barriers are breached by accidental drilling intrusion, the Culebra would provide the most likely pathway for radionuclide transport away from the site. Although water in the Culebra is of poor quality and none is currently used for human consumption, groundwater flow and potential radionuclide transport in the unit have been studied in detail. Results of geologic and hydrogeologic studies of the WIPP region indicate that the geologic and hydrogeologic features of the site will provide effective long-term containment of radionuclides. Geologic and hydrogeologic information is used in the performance assessment that supported the DOE’s compliance certification application to the United States Environmental Protection Agency.