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The omp17.3 gene of Brucella abortus was amplified by PCR and then the amplicon was cloned into the eukaryotic expression plasmid pcDNA3.1 to construct a recombinant plasmid pcDNA3.1-omp17.3. Then the recombinant plasmid pcDNA3.1-omp17.3 was transfected into COS7 cells, and the expressed OMP 17.3 was detected by Western-blotting.

采用PCR方法扩增了布鲁氏菌17.3ku外膜蛋白编码基因，并将该基因克隆至真核表达载体pcDNA3.1中，成功构建了真核表达质粒pcDNA3.1-omp17.3.pcDNA3.1-omp17.3转染COS-7细胞后，通过Western-blotting检测到了17.3ku蛋白的瞬时表达。

Result: 1.(1) Three strips come to appear in the position of 6.0kb, 5.4kb and 600bp after gelose electrophoresis and their size accord with recombined plasmid DNA, pcDNA3 plasmid and VEGF165 gene accordingly.(2) Pure degree of the pcDNA3-VEGF165 recombined plasmid is: OD260/OD280=1.87. Its

在进行了重组质粒的扩增、提取、纯化、鉴定、兔MSCs的原代及传代培养以及RT-PCR、Western Blot 转染检测后，实验取得了令人满意的结果，证实了MSCs 能在体外有效转录外源VEGF 基因并分泌出VEGF 蛋白，同时也说明pcDNA3-VEGF165重组真核表达质粒是有效的。

A eukaryotic expression plasmid composed of the Hst signal peptide sequence in-frame with the human aFGF sequence was used.

有人aFGF序列的信号肽序列在里面结构被使用的Hst的镇静的一真核细胞的表情plasmid。

At the same time, intact shuttle-plasmid pZ189 was also introduced into the host cell which was used for detection of nontargeted mutation frequency. After replicating in cells, progeny plasmid DNA was isolated and transformed into E coli. MBM7070.In the presence of ampicillin, X-gal and IPTG, pZ189-transformed E. Coli MBM7070 forms blue colony if the plasmid contains an active SupF suppresser tRNA gene, and white or light blue colony if the SupF tRNA gene is inactive.

但是由于检测非定标性突变需同时引入穿梭质粒pZ189在细胞中复制，并回收质粒DNA，通过转化宿主菌E.coliMBM7070，在含x-gal和IPTG的氨苄指示板上培养，如果用于检测突变的靶基因SupF tRNA基因发生突变，则菌落色泽为白色或淡蓝色，而正常者为兰色。

The unoperated sides of the treated animals also served as controls. Six normal rats were treated as normal control group. Three different siRNA plasmid solution containing RC2-Ⅰ, MAFbx-Ⅱ, CON (50μl , 0.8μg/μl)was injected and transfected by electroporation as methods mentioned above, respectively. The changes of RC2 and MAFbx mRNA levels and RC2 protein levels after 3 days were determined by real-time quantitative PCR and Western blot, respectively. On postoperative 2, 3 and 4 weeks, the rate of wet muscle weight preservation, mean diameter of muscle fiber and mean cross-section area of muscle fiber and muscle protein content were checked and then compared between group CON and group RC2 or group MAFbx, respectively. The differences between groups were analyzed by one-way ANOVA. Ultrastructural changes of muscle fiber were observed at 2, 3, 4 weeks postoperation.Results GFP plasmid was efficiently deliverd into muscle by electroporation and robust GFP expression in muscle could be observed more than three weeks. Histology shows that injected plasmid DNA diffuses extensively in muscle tissue.

1、健康雌性SD大鼠18只，随机分为电穿孔组和非电穿孔组，每组9只，制作右下肢趾长伸肌失神经支配模型；EP组为将质粒pEGFP-N1溶液50μl(0.8μg／μl)注射入右趾长伸肌后，立即于两侧腱腹交接处给予电穿孔，电穿孔参数为：电场强度为200V／Cm，脉冲100μs，频率1Hz，施加10次脉冲；NEP组仅质粒pEGFP-N1溶液注射；转染后1、2、3周，荧光显微镜下观察趾长伸肌中GFP的表达情况，转染后1周行Western印迹检测趾长伸肌中GFP蛋白的表达情况，检测和优化体内转染效率。2、健康雌性SD大鼠78只，随机分为失神经对照组、RC2基因治疗组(RC2组)，MAFbx基因治疗组，每组24只，制作右下肢趾长伸肌失神经支配模型，余6只为正常组；分别将含CON、RC2、MAFbx基因的siRNA重组质粒注射入趾长伸肌，之后给予电穿孔，方法同上；治疗后3天实时定量PCR和Western印迹检测各组中RC2或MAFbx基因的mRNA和蛋白的表达变化，治疗后2、3、4周检测各组肌湿重维持率、肌细胞直径和肌细胞截面积，肌细胞超微结构变化以及肌纤维中蛋白含量变化。

The CryCI gene and essential regulation elements cut from the plasmid pGF4ABC were inserted into yeast expression vector pPIC9K and plant expression vector pBI 121.1, so we got the secretive yeast expression plasmid pPIC9KBC and plant high-efficiency expression plasmid pGBIF4ABC, Fifty-two His+Muts transformants were obtained after the expression vector pPIC9KBC was introduced into Pichia pasoris, KM7I strain, by electroporation.

将pGF4ABC上的融合杀虫基因CryCI分别克隆到酵母表达载体pPIC9K和植物表达载体pBI121.1上，构建成融合杀虫基因的分泌型酵母表达载体pPIC9KBC和高效植物表达载体pGBIF4ABC。

One was the pqsA' positive expression plasmid constructed by cloning the pqsA'-lacZ fusion digested from pYHP441 into miniCTX-1, whose sequence was then integrated into wild type PAO-1 chromosome by biparental mating procedure. The other was pqsA-E operon knock-out plasmid whose sequence between pqsD and pqsE operon was inserted by tetracycline cassette by the site specific insertion mutagenesis strategy and the mutant constructed by biparental mating of S17-1 that harbored the plasmid and pqsA' positive expression mutant.

一种是酶切质粒pYHP441获得pqsA'-lacZ片段后，亚克隆入质粒miniCTX-1中，构建成PqsA'的阳性表达质粒，随后将构建的质粒，通过双亲交配过程整合入野生型铜绿假单胞菌株PAO-1染色体组中；另一种是通过点特异插入诱变策略，将四环素基因盒插入启动子pqsD和pqsE之间，构建的阴性质粒转化入大肠杆菌S17-1株后，和上述pqsA'阳性表达突变株进行双亲交配过程。

After the NK gene from recombinant plasmid pUC19-NK was cloned on expression vector pBV220, it was transformed into host cell E. coli HB101. Recombinant plasmid pBV220-NK was extracted for identifying by analysis of restriction enzymes, PCR and sequencing. Studies on recombinant strain's growth and expression showed that heterogene have no distinct effect on host cell. The recombinant plasmid have excellent segregational stability but low structural stability. The results of kinetics of pBV220-NK expression in E.

将重组质粒pUC19-NK上的纳豆激酶基因成功克隆到表达载体pBV220上，并将之转入大肠杆菌HB101中，得到转纳豆激酶基因工程菌，提取重组质粒经单酶切、双酶切及PCR分析验证后，对重组菌的生长及表达进行一系列研究，结果表明：外源纳豆激酶基因的导入对宿主的生长没有明显的影响：重组质粒的稳定性实验表明：该质粒具有良好的分离稳定性，而结构稳定性较差。

The full-length genome cDNA containing genetic tags and flanked by Hammerhead ribozyme and Hepatitis delta ribozyme were cloned downstream of the CMV enhancer and the chicken beta actin promoter of the vector pCAGGS. After purified by Plasmid Midi Kit, the recombinant infectious clones named pCAGGmGtAHRT and pCAGGmGtBHRT were directly co-transfected DFI cells indued by Lipofectamine~ 2000. 72h post transfection. the supernatant of cell cuture was harvested and continue passaged in CEF cell. The results of indirect immunofluorescence.

分别在Gt株基因组两端引入了锤头状核酶结构(Hammerhead ribozyme，HamRz)和丁肝病毒核酶结构(Hepatitis delta ribozyme，HdvRz)，并将其克隆在真核表达载体pCAGGS的CMV增强子和β肌动蛋白启动子下游，构建IBDV感染性克隆pCAGGmGtAHRT和pCAGGmGtBHRT，经Plasmid Midi Kit纯化后，在Lipofectamine~ 2000的介导下，直接转染DFI细胞，72h后取上清继续在CEF上传代。

DNA immunization with the Sj16 gene Plasmid DNA was prepared by QIAGEN plasmid mega kit and redissolved in NS at a final concentration of 1μg/μl.

Sj16基因DNA免疫研究用QIAGEN Plasmid mega大量质粒提试剂盒大量制备高纯度的pcDNA〓-Sj16、pcDNA〓和pUC19质粒DNA。

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！