位点特异性重组酶Cre的克隆、表达、纯化及体外活性鉴定

克隆了Cre基因，分别构建了真核表达载体pEF-EBFP-Cre和pGEX-Cre，在大肠杆菌中表达了Cre重组酶，并证明了重组酶的删除特性，为进一步在转基因动物乳腺生物反应器中使用此酶打下基础。     

斑马鱼胚胎中Cre/lox介导的定点整合

采用两种类型的lox突变体和双色荧光报告基因,构建了双交换载体pCMVlox66-CreintEGFPlox2272和Plox71RFPlox2272.将Cre酶基因插入载体pCS2 ,获得了体外转录模板质粒pCSCre.为检测Cre重组酶对斑马鱼胚胎发育的影响,将体外转录的Cre RNA稀释至不同浓度并分别注射入斑马鱼受精卵.结果显示,当Cre RNA浓度高于200ng/μL时会导致高死亡率和高畸形率;而浓度低于100ng/μL的Cre RNA对胚胎发育不造成影响.进一步将50ng/μL的Cre RNA同双交换载体共注射到斑马鱼受精卵中,筛选到发红色荧光的胚胎.PCR检测和测序鉴定证实,在Cre重组酶作用下,两个载体中lox位点间的荧光基因发生了置换反应.实验结果表明,Cre/lox系统介导的盒式交换反应是实现转基因鱼外源基因定点整合的有效策略.     

利用Cre/lox重组系统在转基因动物中筛选高效表达外源基因的"友好"基因座

本实验的目的是利用Cre／lox重组系统进行“友好”基因座的筛选,即利用一个两端带有lox位点的标记基因去转染动物细胞或生产转基因动物,获得可以高效表达外源基因的“友好”基因座。一旦筛选到好的基因座,就建成了一个可以在动物体稳定地高效表达外源基因的技术平台。为此,利用野生型loxP位点和含有一个点突变的lox511位点分别构建两个载体pSL-loxGFP和pSL-loxIFN。首先将含有GFP和新霉素基因的质粒pSL-loxGFP DNA转染牛胎儿成纤维细胞,并用G418筛选,挑选出发绿色荧光的细胞克隆。经增殖培养之后,再将含有鸡γ-干扰素基因的质粒pSL-loxIFN和含有Cre重组酶基因的质粒pBS185 DNA共转染发荧光的细胞克隆,筛选出发生重组后不再发光的细胞克隆。用PCR法检测了两个不发荧光的细胞克隆及发荧光的细胞克隆,并使用质粒pSL-loxIFN、质粒pSL-loxGFP及牛基因组作为对照。检测结果显示,在Cre重组酶的作用下,两个不发荧光的细胞克隆中,一个发生了基因置换,另一个发生了基因删除。以上实验表明,巧妙地用Cm／lox重组系统,可以构建一套使外源基因在动物体内定点地高效表达的技术体系。     

利用Cre/loxP位点特异性重组酶系统从转基因植物生产非转基因食品

转基因植物中的外源基因是引起转基因植物食品安全性问题的主要原因。通过对Cre／loxP系统介导转基因植物中外源基因选择性切除的最新研究成果进行综述分析,指出利用果实、花粉等组织特异性启动子对重组酶基因的表达进行调控,将转基因植物食用部位的外源基因选择性的切除,是将转基因植物转化为非转基因食品的一条有效途径。     

利用Cre/loxP重组系统构建甜菜碱合成酶多基因表达载体

通过使用一套基于Cre/loxP重组系统的植物多基因表达载体构建系统,将辽宁碱蓬(Suaeda liaotungesis kitag)的胆碱单加氧酶(CMO)基因、甜菜碱醛脱氢酶(BADH)基因以及烟草的核基质结合区(MAR)序列构建到同一表达载体上,得到可直接用于农杆菌转化的植物表达载体pYLTAC747H-MAR-BADH-CMO-MAR.该甜菜碱合成酶多基因表达载体的成功构建为进一步进行植物的遗传转化,以有效提高转基因植株的耐盐性提供了实验基础.实验中,用热激法替代了电击法进行质粒的大肠杆菌转化,并去掉了透析等步骤,简化了构建过程.     

N-末端引入RGD序列的葡激酶突变体表达、纯化及性质研究

为解决葡激酶存在的溶栓后再栓塞问题,利用PCR介导的定点突变技术,在野生型葡激酶(wt-SAK)N-末端的第3、4位氨基酸之间插入Arg-Gly-Asp(RGD)序列构建了葡激酶突变体MD2-SAK,另将N-末端的前3位氨基酸替换为RGD序列构建了葡激酶突变体MD4-SAK.将突变体基因分别连接表达载体pBV220,转化大肠杆菌DH5α,经过热激诱导后,突变体均以可溶性形式得到高效表达,表达蛋白占菌体总蛋白的50%以上.破菌上清液通过SP-Sepharose FF、Sephadex G-50和Q-Sepharose FF三步连续的色谱层析纯化得到纤溶活性分别为10.1×104 AU/mg、11.2×104 AU/mg,纯度均大于96%的突变体蛋白.进一步研究了突变体的性质,结果表明葡激酶突变体不仅保留了野生型葡激酶的纤溶酶原激活活性,并具有较强的抗血小板聚集活性.同时发现,葡激酶突变体的N-末端序列会影响其N-末端甲硫氨酸的酶切加工.     

CSF－1R激酶突变子的构建及其突变体在真核细胞的表达

通过ＰＣＲ二步法，构建了ｍＣＳＦ－１Ｒ激酶负性突变子，以及野生型和突变型ＣＳＦ－１Ｒ的送病毒表达载体ｐＣＥＮ／ＭＰＳＶ。进行了１２５Ｉ－ＣＳＦ－１受体结合分析：检测了激酶负性突变子，删除了ＣＳＦ－１ＲＣ－末端氨基酸９２５以后部分的突变体（ＣＴＲＵＮＣ９２５）和删除了激酶插入区的突变体（△ＫＩ）在３２Ｄ髓细胞表达。表达水平可达１～２×１０４受体／细胞。初步测定了通过ＣＳＦ－１Ｒ所介导的促细胞分裂效应。     

互补的烟草花叶病毒介导表达丙型肝炎的核心抗原

ＴＭＶ两种突变体ＴＳＨｃ和ＴＢＤ，共同接种于烟草叶片。Ｎｏｒｔｈｅｒｎ杂交的结果表明这两种突变体由于功能上互补而产生了系统侵染。ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ的检测结果表明烟草的上部叶片表达了丙型肝炎的核心抗原和ＴＭＶ外壳蛋白质。     

基于ajax的电力自动化验收系统设计

在传统基于B／S架构的应用系统中,任何操作都要求重新刷新页面来更新数据,带来比较糟糕的用户体验,而ajax技术可以在很大程度上改善web应用的用户体验,使得B／S架构的应用再也不用让用户陷入不断刷新页面和丢失数据的困局。为了提高系统的开发效率,本系统还使用了三层架构模式进行开发。将三层架构和ajax技术融合于电力自动化验收系统的开发,大大优化了系统的内部结构、提高了系统可维护性、降低系统响应时问还提高了用户体验。     

多功能自动检定系统

文章针对传统手工检定／校准中存在的工作效率低、易出错、信息不能共享等弊端,提出数据处理模板化、系统工作流程化和工作权限分级化的设计方案。系统采用C／S结构与B／S结构相结合的架构模式,应用VB6．0＋ASP3．0＋类C＋VC6．0软件,开发收发管理子系统、检定／校准管理子系统、数据查询子系统、EAM接口子系统和系统维护管理子系统。系统实施后实现了各检定／校准系统的整合集成,从技术上对测量设备检定／校准行为进行规范,优化了测量设备检定／校准流程。该系统符合检定规程及实验室认可标准要求,为测量设备管理流程提供了强有力的支持,实现了检定数据的规范化管理及共享。     

Phase-junction Ag/TiO2 nanocomposite as photocathode for H2 generation

Developing anatase/rutile phase-junction in TiO2 to construct Z-scheme system is quite effective to improve its photoelectrochemical activity.In this work,the anatase/rutile phase-junction Ag/TiO2 nanocomposites are developed as photocathodes for hydrogen production.The optimized Ag/TiO2 nanocomposite achieves a high current density of 1.28 mA cm-2,an incident photon-to-current con-version efficiency(IPCE)of 10.8％,an applied bias photon-to-current efficiency(ABPE)of 0.32 at 390 nm and a charge carriers'lifetime up to 2000s.Such enhancement on photoelectrochemical activity can be attributed to:(i)the generated Z-scheme system in the anatase/rutile phase-junction Ag/TiO2 photocath-ode enhances the separation,diffusion and transformation of electron/hole pairs inside the structure,(ii)Ag nanodots modification in the anatase/rutile phases leading to the tuned band gap with enhanced light absorption and(iii)the formed Schottky barrier after Ag nanodots surface modification provides enough electron traps to avoid the recombination of photogenerated electrons and holes.Our results here sug-gest that developing phase-junction nanocomposite as photocathode will provide a new vision for their enhanced photoelectrochemical generation of hydrogen.     

MOIA: Multi-objective immune algorithm

The paper describes a novel algorithm for finding Pareto optimal solutions to multi-objective optimization problems based on the features of a biological immune system. Inter-relationships within the proposed multi-objective immune algorithm (MOIA) resemble antibody-antigen relationships in terms of specificity, germinal center, and the memory characteristics of adaptive immune responses. Gene fragment recombination and several antibody diversification schemes (including somatic recombination, somatic mutation, gene conversion, gene reversion, gene drift, and nucleotide addition) were incorporated into the MOIA in order to improve the balance between exploitation and exploration. Using five performance metrics, MOIA simulation figures were compared with data derived from a strength Pareto evolutionary algorithm (SPEA). The results indicate that the MOIA outperformed the SPEA in several areas.     

TiO2光催化中价电子的转移过程及其作用

简要分析了二氧化钛光催化中价电子转移过程,即价电子跃迁和电子空穴复合过程,指出价电子转移过程的变化必然引起TiO2光催化效率的变化,重点说明了改变电子受体的种类、半导体表面改性、重金属表面改性、阴离子掺杂和金属离子掺杂等方法的具体原理,进一步探讨了价电子转移过程同光催化效率之间的关系.     

HIV recombination: what is the impact on antiretroviral therapy?

Retroviral recombination is a potential mechanism for the development of multiply drug resistant viral strains but the impact on the clinical outcomes of antiretroviral therapy in HIV-infected patients is unclear. Recombination can favour resistance by combining single-point mutations into a multiply resistant genome but can also hinder resistance by breaking up associations between mutations. Previous analyses, based on population genetic models, have suggested that whether recombination is favoured or hindered depends on the fitness interactions between loci, or epistasis. In this paper, a mathematical model is developed that includes viral dynamics during therapy and shows that population dynamics interact non-trivially with population genetics. The outcome of therapy depends critically on the changes to the frequency of cell co-infection and I review the evidence available. Where recombination does have an effect on therapy, it is always to slow or even halt the emergence of multiply resistant strains. I also find that for patients newly infected with multiply resistant strains, recombination can act to prevent reversion to wild-type virus. The analysis suggests that treatment targeted at multiple parts of the viral life-cycle may be less prone to drug resistance due to the genetic barrier caused by recombination but that, once selected, mutants resistant to such regimens may be better able to persist in the population.     

Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles

Dendritic cells (DCs) are professional antigen presenting cells that induce, sustain, and regulate immune responses. Gene modification of DCs is of particular interest for immunotherapy of diseases where the immunes system has failed or is abnormally regulated, such as in cancer or autoimmune disease. Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA. Among various non-viral vectors, chitosan is considered to be a good candidate for gene delivery system, however, lack of cell specificity and low transfection of chitosan need to be overcome prior to clinical use. In this study, mannosylated chitosan (MC) was prepared to induce the receptor-mediated endocytosis and targeting into antigen presenting cells (APCs), especially DCs having mannose receptors. MC showed great ability to form complexes with DNA and showed suitable physicochemical properties for gene delivery system. It had low cytotoxicity and exhibited much enhanced gene transfer efficiency on the macrophage cell line than chitosan itself. Also, MC/DNA complex was more efficient for transferring IL-12 gene into DCs rather than water-soluble chitosan (WSC)/DNA one, which resulted in better induction of INF-gamma from DCs. Therefore, MC is a promising gene delivery system for repeated administration to maintain sustained gene expression, thereby opening the possibility for immunotherapy.     

Enhanced luminescence of Cu-In-S nanocrystals by surface modification

We have synthesized highly luminescent Cu-In-S nanocrystals by heating the mixture of metal carboxylates and alkylthiol under inert atmosphere. We modified the surface of CIS nanocrystals with zinc carboxylate and subsequent injection of alkylthiol. As a result of the surface modification, highly luminescent CIS@ZnS core/shell nanocrystals were synthesized. The luminescence quantum yield (QY) of best CIS@ZnS nanocrystals was above 50%, which is more than 10 times higher than the initial QY of CIS nanocrystals before surface modification (QY = 3%). Detailed study on the luminescence mechanism implies that etching of the surface of nanocrystals by dissociated carboxylate group (CH3COO-) and formation of epitaxial shell by Zn with sulfur from alkylthiol efficiently removed the surface defects which are major non-radiative recombination sites in semiconductor nanocrystals. In this study, we developed a novel surface modification route for monodispersed highly luminescent Cu-In-S nanocrystals with less toxic and highly stable precursors.     

A measuring system with a recombination chamber for neutron dosimetry around medical accelerators

A measuring system for dosimetry of neutrons generated around medical electron accelerators is proposed. The system consists of an in-phantom tissue-equivalent recombination chamber and associated electronics for automated control and data acquisition. A second ionization chamber serves as a monitor of photon radiation. Two quantities are determined by the recombination chamber--the total absorbed dose and the recombination index of radiation quality. The ambient dose equivalent, H*(10), or neutron absorbed dose in an appropriate phantom, can be then derived from the measured values. Tests of the system showed that a 0.5% dose contribution of neutrons to the absorbed dose of photons could be detected and estimated under laboratory conditions. Preliminary tests at the 15 MV Varian Clinac 2300C/D medical accelerator confirmed that the measuring system could be used under clinical conditions. The H*(10) of the mixed radiation was determined with an accuracy of approximately 10%.     

Luminescent silicon nanoparticles formed in solution

This review focuses on the preparation of Si nanoparticles by wet-chemical routes. The methods described include dispersion from porous silicon, etching and surface functionalization of Si/SiO2 powders and direct chemical reaction of Si precursors. Photoluminescence of silicon nanoparticles can be tuned to cover the whole visible spectrum depending on particle size. The excitonic origin or nature of PL has been generally accepted. Some researchers observed exciton recombination across the direct band-gap, i.e., gamma-gamma transitions, while others evidenced indirect nature of excitonic radiative recombination, which becomes direct in very small particles of 1-2 nm. A large redshift of photoluminescence from these small silicon nanoparticles has been explained by a localized surface states model. Others argue that no localized states are found in the band-gap if a complete oxide shell is formed, and the photoluminescence redshift is due to modification of band-edge states.     

Lipopeptide‐Based Nanosome‐Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size‐controlled lipopeptide‐based nanosome system is reported, derived from the blood‐brain barrier‐permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post‐treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide‐based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR‐mediated transient gene editing applications.