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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。

With genomic DNA of Clostridium stercorarium as a template, a structural gene pel9A encoding pectate lyase was obtained by PCR amplification. The pel9A was cloned into pET28a to recombine an express plasmid pPel9A, and then the plasmid pPel9A was transformed into E. coli BL-21, expressed under the control of T7lac promoter at 37℃.

利用PCR技术从耐热梭状芽孢杆菌中扩增得到产耐热果胶裂解酶的结构基因pel9A，并将其克隆于表达载体pET28a中，最后将此重组质粒转化到受体菌E.coli BL-21中进行表达。

The papA gene was amplified from the pilus adhesive cluster of UPEC132, and then was cloned into plasmid pUC18. The recombinant plasmid was identified and subjected to DNA sequencing.

采用此引物扩增132菌株p菌毛粘附基因群的papA基因，扩增产物克隆入质粒，选取阳性重组质粒进行核苷酸序列分析。

The 5＇coding region of its eDNA was optimized according to the codon bias of Pichia pastoris to make it secretively express in Pichia pastoris with higher performance. Two-step DNA synthsis was used to synthesize the eDNA sequence being optimized of ADTZ. OPT-ADTZ was connected with constitutive plasmid pGAPZaA to construct the reecombinant plasmid pNOA. pNOA was linearized and then transformed into Pichia pastoris GS115. Then codonoptimized ADTZ was constitutively and secretively expressed in Pichia pastoris.

为了使重组蛋白rADTZ能在毕氏酵母中高效分泌表达，根据毕氏酵母密码子的偏好性对rADTZ的5'末端的编码区域进行了优化，利用two-stepDNAsynthsis技术合成出ADTZ优化的基因序列OPT-ADTZ，并与组成型表达载体pGAPZaA连接，构建重组质粒pNOA，线性化pNOA后转化至毕氏酵母GS115中，实现了密码子优化的rADTZ组成型分泌表达。

In addition, the plasmid has been shown to remain unintegrated, or extrachromosomal. Thus, plasmid-based gene therapy by intramuscular injection may be safe, cost-effective and have high patient compliance due to the acceptability of intramuscular injection.

在所有这些转移方法中，裸DNA直接注射法具有：安全可靠（不会引起炎症反应和导致插入突变）、制作工艺简单、操纵简便（与传统的注射方法非常相近）、可重复注射等优点。

The invention discloses a pyruvate oxidase gene derived from viridian aerococcus, recombinant expression plasmid constructed by the gene and an engineering strain of escherichia coli gene transformed by the recombinant expression plasmid.

本发明公开了一种绿色气球菌来源的丙酮酸氧化酶基因AvPyOD，该基因所构建的重组表达质粒，以及该重组表达质粒转化的大肠杆菌基因工程菌株。

The glucoamylase I gene was inserted into the pAC1 plasmid,and a expression plasmid was constructed,then it was used to transform the yeast strain, S.cerevisiae AS2.1364,without auxtrophic marker,The amylolytic tests showed that when this transformant was incubated on plate of YPDS medium containing 1% glucose and 1% starch at 30℃ for 48 hrs zones of starch degradation could be visualized by stained with iodine vapour.

这说明酵母转化子不仅表达了该酶，且有效地分泌到胞外，从而水解培养基中的淀粉，进一步测定酵母转化子48hrs发酵液的葡萄糖淀粉酶活力为8.4u/ml，培养基中的淀粉已完全水解，其生物产量较高。

We composed the rhoa-gene of Wistar rat with the plasmid of pAdTrack-CMV to get the recombinant plasmid. On the basement, we can use the catholyte lipofetamine to transfected the neural stem cells , In the following, we observe the expression of the rho-gene and the geng how to act the motor neuron and NSCs.

本试验用克隆出的大鼠rhoa基因构建到质粒pAdTrack-CMV中，获得重组质粒pAd-rhoa，用阳离子脂质法介导转染到神经干细胞，观察目的基因的表达及其对运动神经元和神经干细胞的作用。

Objective To construct the chimeric plasmid pcDNA3/HBc/B7.1 and detect the expression of the plasmid in vitro.

目的 构建HBV核心蛋白和B7.1分子真核表达嵌合质粒，并检测其在体外的表达。

The range of MIC90 was-10 μg/ml. The susceptibility rates of the strains to other 10 agents were nearly or equal to 0%. Different plasmid profile strains had almost the same antimicrobial susceptibilities. 25 strains (including 18 strains harboring 34-80×106 plasmids) selectively examined to have no self-transmissible conjugative plasmid.

88.6%，MIC90为-10 μg/ml），对卡那霉素等其余10种药物的敏感率很低或不敏感，不同质粒谱菌株对药物的敏感性几乎相同，对包括 18株含34-80×106质粒在内的 25株菌进行接合转移试验，未检出传递性耐药性质粒。

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！