Ribozyme对NPV感染家蚕细胞的保护作用

用自行设计的能专一切割家核多角体病毒即刻早期蛋白ｍＲＮＡ上三个位蹼的三联体ｒｉｂｏｚｙｍｅ－Ｒ４２６（由Ｒ４７，Ｒ２０８和Ｒ６８７三个ｒｉｂｏｚｙｍｅ串联而成），构建了家蚕细胞中瞬时表达的质粒ｐＧｌ２Ｒｚ，为了检测和筛选的方便，在ＢｍＮＰＶＩＥ启动子和Ｒ４２６之间装入了荧光素酶基因。细胞实验的结果表明，Ｂｍ－Ｎ细胞转染３６小时，荧光素酶基因的表达已达最高峰。细胞中抽提的Ｒ４２６的体外切割反应表明，     

用绿色荧光蛋白进行转基因蚕研究

绿色荧光蛋白（ＧｒｅｅｎＦｌｕｏｒｅｓｃｅｎｔＰｒｏｔｅｉｎ,ＧＦＰ）基因用基因枪和压力渗透法导入蚕卵,它的表达由家蚕核多角体病毒（ＢｍＮＰＶ）的即刻早期（ｉｍｍｅｄｉａｔｅｌｙｅａｒｌｙｓｔａｇｅ,ＩＥ）蛋白基因启动子控制。在紫外灯下能观察到少数５龄幼虫身上显示绿色荧光斑点,ＰＣＲ实验证实部分蚕染色体ＤＮＡ中含有ＧＦＰ基因,说明ＧＦＰ基因已整合到这些蚕的染色体ＤＮＡ中并得到了表达     

用绿色荧光蛋白进行转基因蛋研究

绿色荧光蛋白基因用基因枪的渗透法导入蚕卵，它的表达由家蚕核多角体病毒（ＢｍＮＰＶ）的即刻早期蛋白基因启动子控制。在紫外灯下能观察到少数５龄幼虫身上显示绿色荧光斑点，ＰＣＲ实验证实部分蚕染色体ＤＮＡ中含有ＧＦＰ基因，说明了ＧＦＰ基因已整合到这些蚕的染色体ＤＮＡ中并得到了表达。     

内含子对人凝血因子IX活体表达的增强作用

为了研究内含子在活体水平对凝血因子Ⅸ的表达增强作用，首先采用ｌａｃＺ为报告基因，建立了阳离子脂质体ＳＡ介导的活体基因转移方法。将带有凝血因子Ⅸ（ｈＦⅨ）ｃＤＮＡ的质粒ｐＣＭＶＩＸ和带有ｈＦⅨｃＤＮＡ及其内含子的小基因的质粒ｐＣＭＶｉ＇Ⅸ用ＳＡ脂质体包裹，分别经尾静脉注射到Ｂａｌｂ／ｃ鼠体内，ｐＣＭＶＩＸ注射后未在血浆中检测到ｈＦⅨ蛋白；而ｐＣＭＶｉ＇Ⅸ注射后在Ｂａｌｂ／ｃ鼠血浆中检测到ｈＦⅨ蛋白，     

高表达重组人粒细胞集落刺激因子cDNA的中国仓鼠卵巢细胞株的建立

用质粒ｐＥＦ－ＢＯＳ的增强子、肽链延长因子基因的启动子及其第一内含子替换质粒ｐＥＤ－ＧＣＳＦ的增强子，腺病毒主要晚期启动子及杂合内含子。构建成了更高表达质粒ｐＥＦ－ＧＣＳＦ，转染ＣＨＯ－ｄｈｆｒ－细胞，加入并逐渐升高氨甲喋呤的浓度。结果显示：一定范围内，随着ＭＴＸ浓度增高，重组人粒细胞集落刺激因子表达量随之提高；筛选并建立了一株稳定高表达ｒｈＧ－ＣＳＦ ｃＤＮＡ的细胞株，其表达量为５．０￣５．６μ     

文摘

文摘１３，５－二硝基苯甲酸与３，５－二硝基苯甲酸铝盐的分解热研究（英文）Ｃ９２３３１９０３ＲＥＤＤＹＧＯ，ＲＡＶＩＫＵＭＡＲＫＳ（ＩＤＬＣｈｅｍｉｃａｌｓＬｔｄ，Ｂａｎｇａｌｏｒｅ，ＩＮＤ）：Ｅ０３５０ＣＴｈｅｒｍｏｃｈｉｍＡｃｔａ（ＮＬＤ）１９８（...     

CONTENTS OF NEW CARBON MATERIALS OF Vol.16

Ｎｏ .1ＲｅｓｅａｒｃｈａｒｔｉｃｌｅｓＳＴＵＤＹＯＮＴＨＥＭＩＣＲＯＷＡＶＥＡＢＳＯＲＢＩＮＧＰＲＯＰＥＲＴＹＯＦＣＯＭＰＯＳＩＴＥＭＡＴＥＲＩＡＬＣＯＮＴＡＩＮＩＮＧＣＡＲＢＯＮＮＡＮＯＴＵＢＥＳ　ＷＩＴＨＮＩＣＯＡＴＩＮＧＳＨＥＮＺｅｎｇ ｍｉｎ　ＺＨＡＯＤｏｎｇ ｌｉｎ(1) :1………………………………………………………………………ＡＮＥＦＦＥＣＴＩＶＥＭＥＴＨＯＤＦＯＲＰＩＴＣＨＣＯＭＰＯＳＩＴＩＯＮＤＥＳＩＧＮＺＨＩＬｉｎ ｊｉｅ　ＳＯＮＧＪｉｎ ｒｅｎ　ＬＩＵＬａｎｇ(1) :5………………………     

SI—GaAs中的EL2和EL6缺陷团簇相关性研究

用光致电流瞬态谱（ＰＩＴＳ）方法研究了ＬＥＣＳＩ－ＧａＡｓ原生单晶经常规退火和等时快速退火（ＲＴＡ）深能级缺陷的变化。缺陷ＥＬ２（Ｅｃ－０．８２ｅＶ）ＥＬ１２（Ｅｃ－０．７９ｅＶ）表现出类似的ＲＴＡ特性，应属于ＥＬ２缺陷团簇；缺陷ＥＬ６（Ｅｃ－０．３８ｅＶ），ＥＬ８（Ｅｃ－０．２７ｅＶ）和ＥＬ９（Ｅｃ－０．２４ｅＶ０亦具有相似的ＲＴＡ特性，属ＥＬ６缺陷团簇。实验发现，在热退火中，ＥＬ２团簇缺陷密度     

鸡痘病毒282E4株基因组末端非必需区的序列测定与分析

将鸡痘病毒（ｆｏｗｌｐｏｘ ｖｉｒｕｓ，ＦＰＶ）２８２Ｅ４株７．３ｋｂ的ＢａｍＨＩ片段经亚克隆后，获得ｐＵＦａ，ｐＫＳＦｂ，ｐＵＦｃ和ｐＵＦｄ四个重组策粒。采用ＡＢＩ ３７７ＤＮＡ测序仪荧光标记法对上述质粒进行了序列测定，并应用ＤＮＡ ｓｉｓ Ｖ７．０同ＧｅｎＢａｎｋ中已知的ＦＰＶ序列和痘苗病毒的Ｃｏｐｅｎｈａｇｅｎ株的全序列进行同源性比较。     

人骨形态发生蛋白－1（hBMP－1）成熟肽的基因克隆及其在大肠杆菌中的表达

采用ＰＣＲ方法从ＢＭＰ－１的ｃＤＮＡ中克隆了ＢＭＰ－１成熟肽的编码基因，删除了ＢＭＰ－１前体分子Ｎ端的信号肽序列和Ｃ端的其他序列。用ＨｉｎｄⅢ消化１．３Ｋｂ的ＰＣＲ产物，将０．８４和０．４６Ｋｂ两片段分别克隆到ｐＵＣ载体中进ＤＮＡ序列分析。分别酶切回收ＥｃｏＲ－ＨｉｎｄⅢ和ＨｉｎｄⅢ－ＢａｍＨⅠ两目的基因片段，与大肠杆菌表达载体ｐＢＶ－２２０进行退火连接，使插入基因受控于Ｐ＿ＲＰ＿Ｌ启动子。将含有ＢＭＰ－１成熟肽编码基因重组表达质粒ｐＢＶＢＭＰ－１转化大肠杆菌宿主细胞进行温度诱导表达。结果表明，经４２℃热诱导后，大肠杆菌能以包涵体形式表达ＢＭＰ－１成熟肽，其分子量约为５２ｋＤａ。     

Iron‐Oxide‐Based Nanovector for Tumor Targeted siRNA Delivery in an Orthotopic Hepatocellular Carcinoma Xenograft Mouse Model

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Small interfering RNA (siRNA) holds promise as a new class of therapeutics for HCC, as it can achieve sequence‐specific gene knockdown with low cytotoxicity. However, the main challenge in the clinical application of siRNA lies in the lack of effective delivery approaches that need to be highly specific and thus incur low or no systemic toxicity. Here, a nonviral nanoparticle‐based gene carrier is presented that can specifically deliver siRNA to HCC. The nanovector (NP‐siRNA‐GPC3 Ab) is made of an iron oxide core coated with chitosan‐polyethylene glycol (PEG) grafted polyethyleneimine copolymer, which is further functionalized with siRNA and conjugated with a monoclonal antibody (Ab) against human glypican‐3 (GPC3) receptor highly expressed in HCC. A rat RH7777 HCC cell line that coexpresses human GPC3 and firefly luciferase (Luc) is established to evaluate the nanovector. The nanoparticle‐mediated delivery of siRNA against Luc effectively suppresses Luc expression in vitro without notable cytotoxicity. Significantly, NP‐siLuc‐GPC3 Ab administered intravenously in an orthotopic model of HCC is able to specifically bound to tumor and induce remarkable inhibition of Luc expression. The findings demonstrate the potential of using this nanovector for targeted delivery of therapeutic siRNA to HCC.     

Engineered Ferritin Nanoparticles for the Bioluminescence Tracking of Nanodrug Delivery in Cancer

The identification of a highly sensitive method to check the delivery of administered nanodrugs into the tumor cells is a crucial step of preclinical studies aimed to develop new nanoformulated cures, since it allows the real therapeutic potential of these devices to be forecast. In the present work, the ability of an H‐ferritin (HFn) nanocage, already investigated as a powerful tool for cancer therapy thanks to its ability to actively interact with the transferrin receptor 1, to act as an efficient probe for the monitoring of nanodrug delivery to tumors is demonstrated. The final formulation is a bioluminescent nanoparticle, where the luciferin probe is conjugated on nanoparticle surface by means of a disulfide containing linker (Luc‐linker@HFn) which is subjected to glutathione‐induced cyclization in tumor cell cytoplasm. The prolonged imaging of luciferase+ tumor models, demonstrated by an in vitro and an in vivo approach, associated with the prolonged release of luciferin into cancer cells by disulfide bridge reduction, clearly indicates the high efficiency of Luc‐linker@HFn for drug delivery to the tumor tissues.     

Surface‐Tunable Bioluminescence Resonance Energy Transfer via Geometry‐Controlled ZnO Nanorod Coordination

The use of ZnO nanorods (NRs) as an effective coordinator and biosensing platform to create bioluminescence resonance energy transfer (BRET) is reported. Herein, a hydrothermal approach is applied to obtain morphologically controlled ZnO NRs, which are directly bound to luciferase (Luc) and carboxy‐modified quantum dot (QD) acting as a donor–acceptor pair for BRET. BRET efficiency varies significantly with the geometry of ZnO NRs, which modulates the coordination between hexahistidine‐tagged Luc (Luc‐His₆) and QD, owing to the combined effect of the total surface area consisting of (001) and (100) planes and their surface polarities. Unlike typical QD–BRET reactions with metal ions (e.g., zinc ions), a geometry‐controlled ZnO NR platform can facilitate the design of surface‐initiated BRET sensors without being supplemented by copious metal ions: the geometry‐controlled ZnO NR platform can therefore pave the way for nanostructure‐based biosensors with enhanced analytical performance.     

Presence of a biomaterial implant facilitates induction of experimental infective endocarditis due to streptococci and staphylococci

Infective endocarditis (IE) usually is studied using animals with catheters inserted into the heart, which causes formation of platelet-fibrin thrombi (vegetations, VGs). We used two rabbit models to study the respective roles of the catheter and the VGs in the development of IE. The influence of the catheter was studied by either removing the catheter before bacterial challenge, or leaving the catheter in place. In all cases, removal of the catheter caused a strong decrease in the frequency of IE. The presence of the catheter stimulated population increase of streptococci within 4 h after challenge. As most catheters were sterile 4 h after challenge, they did not serve as a reservoir of bacteria. To study the requirement of a preformed VG catheters were inserted either 24 h or 30 min before bacterial challenge. In the former model VGs were present, in the latter VGs were not yet formed when bacteria were injected. The frequencies of IE due to 2 S. sanguis and 2 S.epidermidis strains in the 24 h model or 30 min model were similar, indicating that a preformed VG is not necessary for development of IE. Five coagulase-negative stains were shown to vary in their capacity to cause IE in the 30 min model. Variation was not caused by differences in early adhesion or colonization of the aortic valve, but reflects differences in persistence after initial colonization. Like in the 24 h model, persistence of the bacteria was greatly enhanced by the continuous presence of the catheter. Possible mechanisms of the infection-potentiating effect of the catheters are discussed.     

Preparation of biotinylated cypridina luciferase and its use in bioluminescent enzyme immunoassay

Cypridina luciferase, a well-known secretory enzyme involved in the bioluminescence of marine ostracod, is used to monitor gene expression in mammalian cells. Here we report the preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay (BLEIA). Recombinant Cypridina luciferase was expressed in yeast and successfully purified to near homogeneity. The luciferase was biotinylated with conventional biotinylation reagents, and the biotinylated lysine sites were determined by liquid chromatography-tandem mass spectrometry. The biotinylated luciferase was stable when stored at 4 degrees C. The stability of synthetic S-Cypridina luciferin was significantly improved by adding antioxidants to Tris-HCl buffer. The biotinylated luciferase and S-Cypridina luciferin were used in a model study of the immunoassay for interferon-alpha. The linearity of this immunoassay extended from 7.8 to 500 pg/mL interferon-alpha. Our results show that Cypridina luciferase is a very sensitive and versatile bioluminescent reporter.     

抗菌核病转基因油菜植株的获得

将携带菜豆几丁质酶基因的植物表达载体ｐＢｃｈ通过农秆菌介导法导入甘兰型油菜Ｈ１６５中。经卡那霉素对Ｔ０、Ｔ１和Ｔ２代植株进行连续的筛选和菌核病（ｓｃｌｅｒｏ－ｔｉｎｉａ ｓｃｌｅｒｏｔｉｎｏｒｉｕｍ）接种试验,共获得２２株抗菌核病的Ｔ２代植株。对其中的２２株进行ＰＣＲＳ检测,从６株扩增得到与菜豆几丁质酶基因大小对应的ＤＮＡ带型,Ｓｏｕｔｈｅｒｎ杂交证实其中２株呈现阳性结果,表明外源基因已整合到油菜     

精子干细胞转染法制备转基因兔的研究

采用直接注射的方法 ,将脂质体包裹的含人乳铁蛋白基因重组质粒 pLNCXHLF注入兔睾丸组织中 ,一个月后与正常雌兔交配。所产仔兔经PCR、Southern检测证明获得了转基因兔 ,转基因阳性率平均为 35 %。实验结果表明 ,通过注射可将外源基因导入到曲细精管中 ,进而完成对精子干细胞的转染 ,获得携带外源基因的成熟精子 ,受精后可得到转基因动物。该方法是一种简捷、有效的新途径 ,既节省时间又降低了成本 ,为利用精子载体制备转基因动物的研究提供了很有价值的参考。