The Construction of a New Mobile Recombineering System of pYM-Red

重组工程是近几年发展的新型遗传工程技术. 以PCR扩增的线性低拷贝质粒pACYC184为载体,用Gap-repair方法从大肠杆菌DY330染色体上直接体内亚克隆了包括Red重组酶基因在内的长约6.7 kb的基因序列,构建了pYM-Red重组质粒. 在宿主菌W3110体内进行了染色体上galk基因的敲除,验证了Red重组酶的生物功能,并确定了影响pYM-Red重组效率的诱导时间和线性DNA 片段用量. 在42℃诱导10 min和线性DNA打靶分子浓度为300 ng时,pYM-Red的重组效率可达到大约每4 000个电转存活细胞中有1个重组阳性克隆,分别比pKD46和pBR322-Red系统高5~6倍.Recombineering is a new developed genetic engineering technology in the past few years. A new recombineering system named pYM-Red was constructed by gap-repair, that is a technology called in vivo cloning. Linear PCR fragments that amplified from low copy plasmid pACYC184 were used as gene targeting vector. The length of subcloned DNA sequence including Red gene and a series regulatory sequences are about 6.7 kb. The biology function of Red gene in pYM-Red was tested by gene replacement (galkkan) of W3110 chromosome. Factors that effect recombination efficiency were precisely confirmed. When induced 10 min at 42℃ and using 300 ng linear DNA fragment as targeting molecules, the efficiency of pYM-Red mediated recombination can reach one positive recombination clone per four thousands electroporation survived cells, it is 5~6 folds higher than pBR322-Red and pKD46 recombination system.军队“十五”医药卫生科学基金( 01MA 089).~

Development of a New Recombineering System by Gap Repair

应用Gap-Repair新技术,以pBR322为载体,在λ噬菌体Red重组酶的作用下,通过同源重组直接从大肠杆菌DY330染色体上亚克隆了长度为6.7kb的包含Red重组酶基因的λ噬菌体左向操纵子基因序列。建立了一种能够随意在不同细菌宿主中转移的基于pBR32-2Red的重组工程系统。为了验证pBR32-2Red的生物功能,以大肠杆菌染色体上的galk基因为靶标,用Red介导的单链DNA重组技术敲入T→G单碱基突变,使galk基因内编码第145位氨基酸的密码子由TAT转变成TAG,产生了一个琥珀突变。确定了pBR322Red系统的重组功能。Using lambda phage Red recombinase mediated in vivo homologous recombination system,a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique,and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria.To confirm the recombination functions of pBR322-Red,a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T→G mutation in galK gene on the bacterial chromosome.The result demonstrated that a new λ Red-mediated recombineering system based on pBR322-Red was successfully established.:军队“十五”医药卫生科学基金(编号:01MA089)~

Over-expression of PC-1 Gene Increases Survival Ability of C4-2B Prostate Cancer Cell Line

建立稳定表达外源PC-1基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC-1基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-1基因的真核表达载体pcDNA3·1-PC-1稳定转染C4-2B细胞,Western印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-1基因表达.MTT和软琼脂集落形成能力等一系列方法,研究PC-1基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化.结果表明,PC-1基因的高表达能够诱导雄激素受体(AR)调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX3·1、Jagged1、EphA3、SGEF和NOTCH3等表达发生变化.实验结果初步证明,PC-1基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-1基因表达可调控一些重要信号通路.对PC-1基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.To investigate the potential role of PC-1 gene over-expression on prostate cancer progression, a metastasis and androgen independent prostate cancer cell line C4-2B was used, cell clones constitutively expressing PC-1, and their mock-transfected counterparts were constructed. The expression of ectopic PC-1 was analyzed by Western blot and RT-PCR assay. MTT and colony anchor-independent growth in soft agar were performed to evaluate the effects of PC-1 gene expression on prostate cancer progression. RT-PCR and real-time PCR were used to analyse the expression of differential genes between C4-2B-PC-1 and C4-2B-neo cell lines. Our results showed that stable expression PC-1 induced transcription change of some genes, including PSA,PSMA,NKX3.1,Jagged1, EphA3, SGEF and NOTCH3, which were the androgen receptor, regulated genes and important signal transduction pathway molecules. These observations indicated that PC-1 over-expression might play important role in advanced prostate cancer progression, promote prostate cancer cell androgen independent growth, and may regulate multiple signal transduction pathways. Further study of PC-1 gene function may provide clue to discover new molecular targets for prostate cancer diagnosis and treatment.国家自然科学基金资助(No.30070296)~

Molecular Cloning and Characterization of a Putative Promoter Region of mPC-1 Gene Homologous to hPC-1

为了鉴定鼠mPC-1基因表达的调控元件,克隆并分析了该基因的启动子.构建了一系列mPC-1基因启动子的截短序列.通过荧光素酶报道基因,分析了它们在前列腺癌细胞和其它细胞中的表达.结果表明,在AR阳性细胞系中,mPC-1基因启动子活性远远高于SV40和p61-PSA启动子,mPC-1基因启动子599bp至449bp可能含有一个负调控元件;mPC-11.1kb启动子控制的表达主要在前列腺癌细胞系中;雄激素可调控mPC-11.1kb启动子表达.mPC-11.1kb序列是一个有前列腺癌细胞特异性和较强的启动子,经过进一步的修饰有可能作为一种有用的前列腺癌基因治疗元件.To identify the regulatory region that are responsible for the expression of mPC-1,we have isolated and characterized the mPC-1 gene promoter.Sequence analysis of the mPC-1 5'-flanking region and a series of truncated constructs were performed,which were transiently transfected into the prostate cancer cell lines and non-prostate cancer cell lines and analyzed through Dual-luciferase reporter assay system.The relative activity of mPC-1 gene promoter was by far higher than pGL3-control containing SV40 promoter and enhancer and p61-PSA containing hPSA 6 kb promoter in AR(androgen receptor,AR)-positive prostate cancer cell lines.The region from 599 bp to 449 bp of mPC-1 promoter might contain a negative regulatory element.The expression of mPC-1 1.1 kb fragment is mainly restricted into prostate cancer cell lines.The relative activity of mPC-1 1.1 kb 5'-flanking region was regulated by androgen.The results demonstrated that the 1.1 kb fragment of mPC-1 5'-flanking region was relatively strong and prostate cancer cell specific promoter region.The 1.1 kb promoter of mPC-1 gene might be well suited to prostate cancer gene therapy if the promoter was properly modified.国家高技术研究发展计划(863计划)(No.2002AA223061);; 国家自然科学基金(No.30070296)资助~

Electrochemical Properties of Polymeric Gel Electrolyte with GBL/EC as Plasticizers

以GBL/EC复配体系为增塑剂, PVDF HFP和PMMA为聚合物基体制备胶态聚合物电解质. 研究聚合物电解质的离子传输特性和电化学稳定性. 实验表明,室温离子电导率达到 1. 2mS·cm-1, 电化学稳定窗口在 4. 5V以上. 以GBL/EC增塑聚合物电解质与表面经修饰的锂金属电极组成锂金属聚合物电池,其电极稳定性较好,充放电循环寿命得到很大的提高.Polymeric gel electrolytes (GPE) based on PMMA and PVDF-HFP were prepared using the binary mixtures of γ-butyrolactone (GBL) and ethylene carbonate (EC) as plasticizers. The ionic transport properties and electrochemical stability were studied. The room temperature conductivity reached 1.2 mS·cm~(-1), electrochemical stability window exceeded 4.5V. The interfacial stability between GPE and lithium electrode was also investigated. The lithium metal polymer battery (LMPB) prepared with the GPE showed better charge and discharge life cycle when the the lithium metal electrode was treated with 1,4-dioxane.作者联系地址：哈尔滨工业大学理学院应用化学系,哈尔滨工业大学理学院应用化学系,广东省江门三捷电池实业有限公司研究中心,广东省江门三捷电池实业有限公司研究中心,哈尔滨工业大学理学院应用化学系,哈尔滨工业大学理学院应用化学系 黑龙江哈尔滨150001广东省江门三捷电池实业有限公司研究中心广东江门529000 ,黑龙江哈尔滨150001 ,广东江门529000 ,广东江门529000 ,黑龙江哈尔滨150001 ,黑龙江哈尔滨150001Author's Address: 1. Department of Applied Chemistry, Harbin Institute of Technology,Harbin 150001,China,2.Research center of JJJ Battery Co. LTD.,Jiangmen 529000, Chin