质粒

On the basis of our results,IFN-θnot only has the sequence homology to other typeⅠinterferons,but also matches the primary properties of typeⅠinterferons in antiviral, IFNαR receptor binding and virus inducing,etc.

首先，将PoIFN-α基因克隆入pcDNA3真核表达载体中，筛选阳性克隆后大量抽提质粒，通过脱水化-再水化步骤将重组质粒包裹入阳离子脂质体内制备成核酸佐剂(IFN-adjuvant)。

pPR2 is the smallest ac-tinomycete linear plasmid beyond Streptomyces. This is the first time to report the complete nucleotide sequence of linear plasmid in the genus Planobispora. pPR2 may have novel mechanism for telomere replication, and pPR2.2c and pPR2.3c encode the possible telomere replication proteins.

pPR2是链霉菌之外的放线菌中最小的线型质粒，其序列在游动双孢菌属的线型质粒中是首次报道。pPR2可能具有新型的端粒复制的机制，其中pPR2.2c和pPR2.3c编码可能的端粒复制蛋白。

Methods: Two recombinant palter plasmid containing whole CDNA sequences of HMCD59 transfected CHO cells by lipofectamine with PCDNA respectively. Then G418 was used to select positive clone.

分别构建两种含有HMCD59全长CDNA序列的重组pALTER质粒，并与pCDNA3质粒，按3：1比例混合后，运用脂质体介导法共转染CHO细胞(编号为HM5-CHO及HM6-CHO)，用新霉素类似物G418初步筛选出阳性克隆。

Results:Mutant CD59 cDNAs subcloned into the mammalian expression vector PLATER and transfected CHO together with the pcDNA,which confered resistance to G418. The positive clones were tested by FIH.

结果：运用阳离子脂质体导入法将重组pALTER质粒与pcDNA3质粒共转染CHO，成功筛选出的阳性克隆经FIH证明突变CD59可在CHO细胞表达。

However, there are still several safety concerns about the use of a DNA vaccine, which include the possibility of integration into the host genome, adverse immunopathology, and anti-DNA autoantibody induction.Attempting to solve some of these problems, eukaryotic expression plasmids pTL-8/ AMA-1 and pTL-8/ AMA-1 which express trans-activator or reverse trans-activator, respectively, and AMA-1 gene of Plasmodium falciparum were constructed by using tetracycline regulable system.

首先构建了恶性疟原虫顶第四军医大学硕士学位论文端膜抗原1(AMA一1)基因和反式活化因子基因的真核表达质粒pTL一8/灿认一l和PTL一8/灿认一1，并大量制备这两种质粒及表达转录抑制子的质粒pUHS6一1。

Expression of M, NP, F and HN genes were detected and confirmed by the indirect immunofluorescence analysis. The syncytium formation was observed in the HeLa cells co-transfected with pCAGG-M, pCAGG-NP, pCAGG-F and pCAGG-HN recombinant plasmids at 48h post transfection.

纯化后的重组质粒通过脂质体单独转染HeLa细胞，经间接免疫荧光试验检测到了M、NP、F和HN蛋白的表达。pCAGG-M、 pCAGG-NP、pCAGG-F和pCAGG-HN重组质粒组合共转染HeLa细胞48h后，可以观察到明显的细胞融合现象。

Heteroduplex molecules have been made by mixing pJSA1175 and pJSAl175ADRV4, pJSA1175ADRV4 and pJSAl175RRV4 DNA molecules which have been linearized by Pst Ⅰ and Bgl Ⅱ respectively, denaturing them in alkaline or high temperature, allowing them to renature and then spreading in the presence of formamide.

以痘苗病毒表达载体pJSA1175及重组质粒pJSA1175ADRV4(含ADRV VP4基因)、pJSA1175RRV4(含猴轮状病毒VP4基因)为材料，用碱变性方法或热变性方法制备了载体质粒与重组质粒DNA之间的异源双链，电镜下成功观察到载体序列部分经变性、复性后配对呈双链状态，而外源基因区域突出呈单链环状结构，测量长度为2.3kb。

The plasmid pcDNA3.1-3-1E and pcDNA3.1 was respectively used to immunize SPF chicks by injection intra-muscularly in thigh muscle at 14 and 21 days of age.Chicks in each group except that in the unchallenged control group were challenged with 5×104 E.acervulina sporulated oocysts at 28 days post-inoculation,and the immune protective efficacy was observed.

将构建的重组质粒以及空载体质粒分别于14、21日龄分2次经腿部肌肉注射免疫SPF雏鸡，28日龄除非免疫非感染对照组外各组攻击性感染5×104个堆形艾美球虫孢子化卵囊，观察真核表达质粒对球虫感染鸡的免疫保护作用并探讨其免疫保护机理。

Results were shown as followings:(1) Sodium selenite at 0～2.5 μmol/L significantly increased the antioxidative capacity of L-02 cells without having remarkable impact on SMMC-7721 cells;(2) Sodium selenite at concentrations above significantly increased telomerase activity, hTERT gene expression and telomere length of L-02 cells without significant impact on SMMC-7721 cells;(3) Sodium selenite at higher concentrations (larger than 5 μmol/L) resulted in peroxidation of L-02 cells, while scutellarin significantly counteracted its effect;(4) Selenium-rich amino acids from silkworm pupas in the range of 0.5～2.5 μmol/L Se significantly inhibited SMMC-7721 cell growth, induced apoptosis and cell cycle change, and the generation of reactive oxygen species. In contrast, sodium selenite and selenomethionine only had weak impact on them at the same concentrations;(5) A new selenium-containing protein was found from selenium-rich silkworm pupas, which is worthy to be study further;(6) An expression vector containing ansense RNA of hTERT gene were constructed and used to transfect SMMC-7721 cells. They were observed to inhibit hepatoma cells.

结果如下：（1）0～2.5μmol/L亚硒酸钠显著性增强L-02细胞的抗氧化能力；而对SMMC-7721细胞的作用不显著；（2）该浓度硒显著性提高L-02细胞端粒酶活性、增强hTERT基因表达和延长细胞端粒长度；而对SMMC-7721细胞的作用均不显著；（3）高浓度硒（5μmol/L以上）显著性抑制L-02细胞生长、致细胞过氧化，灯盏花素能拮抗硒所致过氧化、降低硒毒性；（4）0.5～2.5μmol/L富硒蚕蛹氨基酸显著性抑制肝癌细胞SMMC-7721生长、导致细胞凋亡和周期改变、诱导细胞产生活性氧，同浓度亚硒酸钠和硒代蛋氨酸对其抑制不显著；（5）富硒蚕蛹蛋白经分离纯化和鉴定后发现存在一新含硒蛋白，其结构和功能有待研究；（6）通过已有的含hTERT基因质粒，成功构建hTERT反义RNA表达质粒，转染SMMC-7721细胞后对其生长具有抑制作用。

All index proved that the P5776 cDNA library constructed in this research had high quanlity and could be used for further ESTs sequencing and analyzing. The amplified library of P5776 was screened on white-blue plates, and more than 5,000 white-clones were selected to culture in 96-well plates, then the high quanlity plasmids of white-clones were extracted by traditioal alkaline-lysis method using the Vitagene?96-easy plasmid extract kit. Through PCR primered by T3( the vector has T3 RNA polymerase promoter ), the target fragments of inserts were get. Then the inserts were taged by four different fluorescence-dye, and fractionated by capillary in ABI Prism? sequencer. More than 5,000 5′-ESTs had been sequenced and through rough selecting, 4,747 ESTs were remained for further research. Among the remained ESTs, more than 90% have longer lenth than 500bp, and there are less than 5% unable-read bases in every sequence.

将扩增好的P5776 cDNA文库铺布蓝白斑筛选平板，挑取其中的白斑经活化培养后，运用Vitagene 96-easy质粒DNA制备试剂盒以碱裂解法共提取出58 板(96孔板)，即5，000 余个重组子克隆的高质量质粒DNA；提取好的质粒以T3(连接载体上具有该引物RNA聚合酶启动子)为引物经PCR(Polymerase Chain Reaction，聚合酶链式反应)扩增出重组子插入片段，标记上荧光染料后在ABI Prism 3100 测序仪上经毛细管电泳完成了5，000 余条5′端EST 序列的测定工作，初步筛选出4，747 条质量较好的EST序列，经筛选得到的EST中90%以上具有大于500bp 的可读序列，每一EST序列中不可读碱基数小于5%。

She gently rebuff ed him, but agreed that they could be friends

她婉言拒绝了，但同意作为朋友相处。

If in the penal farm, you were sure to be criticized.

要是在劳改农场，你等着挨绳子吧！