胞质基因

Methods S. epidermidis atlE gene expression level at different phases was detected by RT-PCR, and the accumulation of extracellular DNA at different phases was assessed by spectrophotometric measurements of light absorbance by DNA; DNase Ⅰ was used to study the function of extracellular DNA in biofilm formation and primary attachment. The effects of atlE gene deletion on initial adherent capacity, biofilm formation and extracellular DNA release were studied by construction of atlE deletion mutant via homologous recombination.

采用RT-PCR法检测表皮葡萄球菌atZE基因在不同时期的表达水平，用紫外分光光度计检测DNA的方法检测了相应时期的胞外DNA的释放量，并用DNA酶研究表皮葡萄球菌胞外DNA在生物膜形成和起始黏附中的作用；采用ρBT2质粒同源重组敲除的方法构建了表皮葡萄球菌1457的atlE基因突变株，研究atlE基因敲除突变对起始黏附能力、生物膜形成及胞外DNA释放能力的影响。

Methods Westernblot was employed to detect the intracellular expressions of caspase-9, hTERT, Bcl-2 and Bax, and mitochondrial and cytoplastic cytochrome C in HepG2 cells transfected by pSliencer 3.1-H1 neo-shTERT.

收集第20代hTERT基因RNAi真核表达载体论定转染细胞、对照细胞及未转染HepG2细饱，采用Western blot检测线粒体，胞质细胞色素C的表达及细胞内caspase-9、Bcl-2、Bax和hTERT的表达。

Drug sensitive test and three-dimensional test220 strains of Pa were isolated from hospitalized patients between 2003 and 2007. K-B method was used to tested the susceptibility of 10 different antibiotics. IRPa was screened by testing the minimal inhibitory concentration of imipemem by using agar diluiion method.The susceptibility of these IRPa to the antibiotics was analysised. Three-dimensional test was used to identify the different kinds of beta lactamases from 220 strains of Pa.2.Carbarpenems hydrolytic enzyme genes and oprD2 gene were detectedamong the selected IRPa strains, PCR method was performed to detect carbapenemase genes which included GES、KPC、SPM、VIM、IMP、GIM gene and the oprD2 gene;Multiplex PCR were used to detect OXA genes and plasmid-mediated AmpC beta lactamase genes; The expression of the chromosomal AmpC beta lactamases and oprD2 genes in IRPa strains were analyzed by Real-time PCR.3.Identification and characterization of integronsIntegrase gene was detected by PCR, and the classification of integrons was performed by using restriction fragment length polymorphism.PCR was performed to detect the qacE△1-sull gene,and the gene cassetes which are located at variable region of integrons in the strains were detected to be positive.

方法1、药敏实验和三维实验收集2003～2007年临床分离的220株Pa，对这些菌株采用K-B法测定10种临床常用抗生素的药敏情况，同时采用琼脂稀释法检测亚胺培南的最低抑菌浓度(Minimal inhibitory concentration，MIC)，筛选出对亚胺培南耐药的铜绿假单胞菌，并分析其对其它抗生素的药物敏感率；采用三维实验的方法分析220株Pa产β内酰胺酶的类型。2、碳青霉烯类水解酶和oprD2蛋白的检测针对鉴定的IRPa菌株，采用普通PCR方法检测具有碳青霉烯水解作用的β内酰胺酶耐药基因(GES、KPC、SPM、VIM、IMP、GIM基因)和oprD2基因，采用多重PCR的方法检测OXA型基因和质粒携带的AmpC酶基因，用荧光定量RT-PCR方法检测oprD2蛋白基因表达情况；同时对产AmpC酶的Pa(25株，含IMP耐药和敏感株)用RT-PCR方法检测AmpC酶基因的表达量情况。3。

The construction process includes the following steps: constructing recombinant plasmid containing phhA and phhB; transforming the recombinant plasmid to hygrophilous aeromonad; and screening recombinant hygrophilous aeromonad with the recombinant plasmid.

该重组菌中含有β-酮硫解酶基因phbA和乙酰乙酰辅酶A还原酶基因phbB。其构建方法包括以下步骤：1构建含phbA和phbB基因的重组质粒；2转化该重组质粒到嗜水气单胞菌中；3筛选出含有重组质粒的重组嗜水气单胞菌。

Glioma is still one of refractory disease in the neurosurgical field; the development of new primary and adjuvant treatment is vital. Recently, the gene therapy of glioma is developed rapidly and there are many methods about the gene therapy that include: suicide gene therapy, immunologic gene therapy, drug resistangce gene therapy, angiostatin gene therapy and so on. The sucide gene therapy is the most potential approach of antitumer, these nonmammalian genes encode enzyme that convert nontoxic prodrugs into highly toxic metablites. Cells transfected with suicide genes are targeted for specific negative selection, witch can be induced by administrtion of the corresponding produg. Among the enzyme/produg combinations, two of the best characterized system are herpes simplex virus thymidine kinase /ganciclovir and Escherichia coli cytosine deaminase /5-flourocytosine (5-FC). The formor can convert the antiviral nucleoside analogs acyclovir , ganciclovir to their nucleoside monophosphate derivatives, the monophosphate forms are subsequently phosphorylated by endogenus cellular kinases to triphosphates, these molecules are potent inhibitors of DNA synthesis.

近年来脑胶质瘤的基因治疗发展迅速，应运而生的方法有自杀基因、免疫基因、多药耐药基因以及抗血管生成基因等，其中自杀基因被认为是最有前景的基因治疗方法，它又称病毒介导的酶／药物前体疗法，是利用转基因技术将哺乳动物细胞中所不含有的自杀基因转入到哺乳动物肿瘤细胞中，该基因表达的产物可将无毒的药物前体转化为毒性药物，从而选择性杀伤该肿瘤细胞，常用的自杀基因有单纯疱疹病毒-胸苷激酶基因和大肠杆菌胞嘧啶脱氨酶基因，前者催化无毒性抗病毒核苷类似物如丙氧鸟苷、无环鸟苷等成为单磷酸核苷衍生物，然后在内源性细胞激酶作用下转化为具有明显毒性的三磷酸核苷，作为DNA合成链的终止剂和DNA合成酶的抑制剂，干扰细胞DNA的合成；后者编码的胞嘧啶脱氨酶可催化5-氟胞嘧啶（5-FC）脱氨成为5-氟尿嘧啶（5-FU），然后代谢为有毒性的5-氟尿嘧啶-5′三磷酸（5-FUTP）和5-氟-2′脱氧尿嘧啶-5′磷酸（5-FdUTP），5-FUTP通过与UTP竞争性结合而抑制mRNA和tRNA的合成，5-FdUTP则作用于胸苷合成酶，导致TMP衰竭而阻止DNA的合成，最终诱导肿瘤细胞凋亡。

Comparison from the Germany isolate, phocine distemper virus type Ⅱ(PDV-2) and vaccine strain, the major difference is the long signal peptide domain while the mature protein exhibit high identity, and all of the 13 serine residues, four potential asparagine glycosylation sites and two hydrophobic regions supposed to affect the fusion function are completely identical. These data supports the view that F protein is more conservative than H protein in CDV.

三、犬瘟热病毒中国分离株囊膜糖蛋白基因免疫小鼠诱发的抗体应答经一系列步骤将CDV-YZ0101株的两囊膜糖蛋白基因定向导入真核表达载体pcDNA3.1中，DNA测序和酶切分析筛选阳性重组质粒克隆pcDNA-H和pcDNA-F，以重组质粒DNA和脂质体共转染COS-7细胞，用间接免疫荧光试验证实转染的COS-7细胞的胞浆中分别表达了CDV的H和F蛋白。

Pombe Yeast is a model organism for studying eukaryote, especially in the research of humam genomics.

因此，依地福新抑制粟酒裂殖酵母胞质分裂可能与MAPK级联相关的mid2、pmp1、spm1基因有关。

The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

We biopsied 1-2 single blastomere from 6-8 cell cleavage stage intracytoplasmic sperm injection embryo, and using nested PCR amplified the high frequently mutation region G380R of FGFR3 gene of the single blastomere. The products of PCR were digested by restriction enzyme Bfm Ⅰ, then the digested products were detected by 10% PAGE to see whether the embryo inherted the mutation of the patient and to screen out normal embryo transfer.

活检经胞质内单精子注射(intracytoplasmic sperm injection，ICSI)授精的胚胎发育至6～8细胞期的1～2个单卵裂球，采用巢式PCR扩增单卵裂球的FGFR3基因的高发突变位点G380R区域，用限制性内切酶BfmⅠ消化巢式PCR的内扩增产物，再经10%聚丙烯酰胺凝胶电泳检测有无遗传患者的该种突变，从而筛选出正常胚胎移植。

That shows there are some difference in the steroid regulation between the anuras and the urodeles. Among three of the steroid, one can convert to the other, and some enzyme activity is concerned with the conversion.7. For the Rana quadranus and the Batrachuperus tibetanus, the steroid receptors play key roles in the synthesis and secretion of the steroids and during the development of the follicle cell.

类固醇激素受体在卵母细胞中可能通过两种途径发挥作用，一种是存在于胞质中，作为信号转导因子的非基因组调控机制；另一种是存在于核中，作为核转录活性因于，改变基因转录活动的基因组调控机制。

Breath, muscle contraction of the buttocks; arch body, as far as possible to hold his head, right leg straight towards the ceiling (peg-leg knee in order to avoid muscle tension).

呼气，收缩臀部肌肉；拱起身体，尽量抬起头来，右腿伸直朝向天花板（膝微屈，以避免肌肉紧张）。

The cost of moving grain food products was unchanged from May, but year over year are up 8%.

粮食产品的运输费用与5月份相比没有变化，但却比去年同期高8％。