质粒

Methods: Genes of MCP and DAF were connected by 10aa coding sequence of enriching Gly and Ser. Then insert into pcDAN3.1 vector, constituting pcDNA-MD3 expression plasmid of hMCP-DAF fusion gene. The genes were transformed by liposome into the pig endothelial cell; human serum handles masculine cloning, identify the function that restrain breaking of person alexin.

将MCP、DAF两个基因以富含Gly和Ser的10aa编码序列作铰链串接，并接入表达载体pcDNA 3.1中，构成含hMCP-DAF的真核表达质粒(pcDNA-MD3)；以脂质体转染猪内皮细胞，人血清处理阳性克隆细胞，鉴定抑制人补体溶破的功能；应用显微注射将线性基因导入猪受精卵；PCR检测导入基因整合率。

GFP gene eukaryotic express plasmids were transfected into ES cells. The cells were observed under the inverted fluorescent microscope. The clones which expressed the powerful green fluorescence were chosen to be named ES-GFP cells and cultured continuously. The examination of ES cell totipotent including Alkine phosphatase staining, embryonic body formation in vitro and teratomas formation in nude mice was implemented.

1。采用脂质体方法将GFP质粒转染ES-D3细胞株，经筛选后倒置荧光显微镜观察细胞形态及荧光状态，挑取荧光最强的克隆，命名为ES-GFP细胞，进一步扩增培养，观察细胞生长及荧光表达情况，行细胞分化全能性鉴定包括碱性磷酸酶（alkine Phosphtase，AKP）染色、体外胚胎体（embryonic body，EB）形成实验及裸鼠体内成瘤实验。2。

The constructed pcDNA3-IL-15 plasmid was identified by Bgl Ⅱ digestion,PCR amplification and sequenceanalysis respectively,to ensure that the hIL-15 cDNA was properly inserted into thevector and the sequence was correct.Then the pcDNA3-IL-15 plasmid was instantlyexpressed in COS-7 cells by means of lipofectamine transfection,and followed by astable expression in Chinese hamster oval cells.8 positive cell colonies wereobtained after the G418 selection,designated CHOA~H cells.

经过BglⅡ酶解分析，PCR扩增鉴定和DNA测序分析，证明IL-15 cDNA已正确插入克隆载体且序列正确后，采用脂质体法，首先将pcDNA3-IL-15重组质粒转染至非洲绿猴肾细胞（COS-7）做瞬时表达，再转染至中国仓鼠卵巢细胞进行稳定表达。

AIM:To assess the antioxidative activity of a new cyclic diaryheptanoid (compound 1) from ginger on free radical damage. METHODS:Hemolysis, MDA levels of mice liver tissue homogenates, the conformation changes of irradiated PUC18 were used as indices.

目的和方法：采用溶血实验，小鼠肝组织脂质过氧化物测定，采用受辐射质粒构象改变等方法测定从生姜中分离得到的一新的环状二苯基庚烷类化合物(化合物1)的抗氧自由基损伤活性。

By the electropositive Liposome 2000, in the 6-plate for cell culture, pEGFP-P80 above is transfected into the PK-15 cells from the kidney of pigs and expressed.

利用阳性脂质体L2000 介导，在六孔细胞培养板中，将已经构建好的pEGFP-P80重组表达质粒转染到PK-15 细胞中进行表达。

Methods Eukaryotic expression plasmid pCMVCD was constructed , and identified by re2 striction endoenzyme digestion. CD gene was transfected into NSCs from new2born Wistar rats using Lipofec2tamine2000. Positive clones (named NSCs/ CD cells) were screened by G418 presence. 52Fluorocytosine (52FC) ad2ministration of different concentrations were incubated with NSCs/ CD cells. NSCs/ CD cells viability ratios were mea2 sured by MTT assay.

通过构建真核表达质粒pCMVCD ，限制性内切酶消化鉴定后，采用Lipofectamine 2000 脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stemcells ， NSCs)，G418 筛选阳性克隆，加入不同浓度的52氟胞嘧啶(52Flourocytosine ， 52FC)，MTT比色法测定NSCs的生存率。

Interval observations under thefluorescent microscop were performed with excition waves of 470—490 nm.The results displayed that GFP genes were expressed effectively in E.octocarinatus, and GFP was distributed unifimly around macronuclearand diffused to the entire cell gradually. The chromosomes can be kept inthe macronucleus for 90h.

用优化的脂质体转化方法将含有人工染色体的重组质粒pBTub-tel2转化到处于有性生殖分裂间期阶段的游仆虫细胞中，结果表明，GFP基因在游仆虫细胞中得以高效表达，36-48小时绿色荧光均匀分布在细胞核周围，逐渐扩散到整个细胞，染色体能够在大核中保持约90小时。

AIM: To establish recombination plasmid pEGFP-NGB and to investigate the expression of pEGFP-NGB in culture neuroglia cells.

目的：构建重组小鼠质粒pEGFP-NGB并研究其在培养神经胶质细胞中的表达。

The expression of CD59 on the cell membrane was tested by cell immunohistochemistry and flow cytometry. Results pALTER plasmid containing CD59 was cut with restriction enzymes and a 496 bp fragment obtained by electrophoresis, which was complete conformity with the insert.

将两种含有不同突变体的人CD59全长cDNA序列重组pALTER质粒，应用阳离子脂质体导入法与pcDNA共转染CHO细胞，用G418筛选阳性克隆，应用细胞免疫组化及流式细胞仪检测CD59在CHO细胞表面的表达。

RESULTS A eukaryotic expression system for high expression humanmutantCD59 were successfully set up : The recombinant PALTER-MAX plasmid containing human mutantCD59 cDNA and PCDNA plasmid were co-transfected into CHO cell by cation lipoid mediating method ;and the cells were grown in F12 medium containing 400ug/ml G418 for 14 days, positive clones were grown in RPMI1640 medium to get stable expressing cell lines . Highly expressing clones were selected by flow cytometry ,and were named PALTER-CD59-CHO1PALTER-CD59-CHO2 . Flow cytometry indicated that expression rates of PALTER-CD59-CHO1 and PALTER-CD59-CHO2 were 53.7%and 54.5%. Further more, Stable highly expressing CHO cell lines were more detected by immunocytochemistry and immunofluorescence technology . PALTER-CD59 -CHO1 and PALTER-CD59-CHO2 were grown in RPMI1640 to get a large of cells . CD59 protein were obtained by spalling PALTER- CD59- CHO1 and PALTER - CD59 - CHO2 cells . Stable highly expressing cells were further validated by SDS-PAGE, immunoblot analysis and solid enzyme immunoassay . PALTER - CD59 - CHO1 and PALTER - CD59 - CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER-CD59-CHO1 or PALTER-CD59-CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59 -CHOI or PALTER-CD59-CHO2 was higher than unglycated ones . PALTER -CD59-CHO1 and PALTER -CD59 -CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER - CD59 - CHOI or PALTER - CD59 - CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59-CHO1 or PALTER - CD5 9-CHO2 was higher than unglycated ones .

结果 成功构建突变人CD59的真核细胞表达系统：运用阳离子脂质体介导法将含有突变人CD59的PALTER—MAX重组质粒与PCDNA共转染入CHO细胞：用含有400ug／mlG418的F12培养基培养14天，筛选出稳定阳性表达克隆，RPMI1640培养基扩增获得稳定表达细胞株，并用流式细胞术进一步筛选出高效表达细胞株分别命名为PALTER—CD59—CH01、PALTER—CD59—CH02，表达率分别为53.7％、54.5％；应用免疫组化方法、免疫荧光技术进一步鉴定阳性细胞株；RPMI1640培养基大量扩增PALTER—CD59—CH01、PALTER—CD59—CH02细胞株，裂解细胞得到CD59蛋白质；通过SDS—PAGE凝胶电泳技术、免疫印迹技术、固相酶联免疫吸附试验验证了这两中文摘要个阳性细胞株CO59蛋白的高效表达；50mM核糖培养72小时，获得突变人CD59糖化细胞株，BCECF染料释放试验结果显示，PALTER一CD59一CHOI、pALTER一CD59一CHOZ细胞较PALTER一CHO细胞染料释放率低，未糖化PALTER一CD59一CHOI、PALTER一CD59一CHOZ细胞比较糖化后细胞染料释放率低。

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！