质粒

PUC19 plasmid were successfully enriched and purified from the E. coli DH5α under the magnetic fields. The obtained plasmid retained native the bioactivity to be used for restriction enzyme digestions and cell transformations.

用这种方法成功地从大肠杆菌DH5α浓缩和纯化得到了pUC19质粒，该质粒具有生物活性，可直接用于限制性酶切和细胞转化等分子生物学下游操作。

The amplified fragment was inserted into pRSET-A to generate the first double repeat of the precursor gene. By utilizing a pair of isocaudamer BamH I and Bgl II sites, and another downstream Hind III site of plasmid pRSET-A, following a series of simple double digestions and ligation of the resulted products, a series of repeat (3, 4 and 6) precursor peptide fragment genes were derived.

本实验设计一对两侧含编码疏水性氨基酸密码子的引物，经过扩增前导序列10~34aa基因序列，并重新克隆入质粒pRSET-A构建串联二聚体后，再利用质粒pRSET-A的BamH I / Bgl II同尾酶克隆位点，经一系列简单的酶切和连接，快速构建这一前导肽中不含组氨酸标签序列的串联多聚体基因，并成功表达其六聚体重组蛋白。

Full length LMP2A cDNA was firstly incised from pGEM-T-LMP2A with EcoR Ⅰ, Sma Ⅰ digestion, and then inserted into eucaryote. expression plasmid pCIcc controlled by CMV promoter. The CMV-LMP2A-SV40 expression unit was digested by ClaI, and inserted into E1-substituted adenovirus vector pAx1cw. Then the LMP2A recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22I digested Ad5-TPC.

将带有LMP2A cDNA的重组质粒pGEM-T-LMP2A用EcoR Ⅰ、Sma Ⅰ双酶切下LMP2A cDNA，并将其插入含同样酶切位点的真核表达质粒pCIcc中，使其受控于CMV启动子下；用ClaI切下CMV-LMP2A-SV40表达单元，插入E1、E3区替代的腺病毒载体pAX1CW，选择正确的克隆pAX1CW-LMP2A与Ad5 DNA-末端肽复合体共转染293细胞，通过同源重组获得复制缺陷型的重组腺病毒(Ad5-LMP2A)。

Methods The immunofluorescency, Western blotting and RT-PCR were performed on mouse liver tissue obtained from 4 experimens pcDNA3.1(+-HBX and 1 normal control, 24 h after delivery the HBX gene eukaryon expression vector pcDNA3.1-HBX into mouse by the means of hydrodynamic injection.

实验动物分成模型组和对照组，模型组利用已构建的含有HBX基因的真核表达质粒pcDNA3.1-HBX，对照组以等量生理盐水代替，采用流体动力学法将质粒经尾静脉高压注入小鼠体内，24h后取小鼠肝组织，行免疫荧光、RT-PCR和Western blot法从不同水平检测HBX在小鼠肝组织内的表达情况。

The minor core protein s C-encoding gene of Muscovy duck reovirus was cloned into theprokaryotic expression vector pET32a. The recombinant plasmid pET32a-s C was amplified andextracted after being transformed into E.coli DH5a competent cells. Restriction analysis withEcoRⅠand SacⅠand sequences analysis indicated that the recombinant plasmid was inserted withcorrect open reading frame. The fusion protein about 50 ku was produced after induction with 0.15mmol/L IPTG of E.coli competent cells transformed with pET32a-sC. The SDS-PAGE andWestern-bloting test indicated that the fusion protein reacted with the convalescents sera of duckinfected with Muscovy duck reovirus. The indirect ELISA method was developed by using thepurified fusion s C protein as coating antigen. The optimal concentration of s C was 5μg/ml, thedilution of serum sample was 1:40; The results showed that preparation of an ELISA by using sCas coating antigen in detecting 50 field duck sera in comparison with the AGIP were more sensitiveand specific than agar gel immuno-diffusion AGIP test. The results suggest that presence ofantibody against viral protein sC in duck may be a good indicator by the sC-ELISA for detectionof duck infection with reovirus.

同时，本研究将编码外壳蛋白σC的基因克隆于原核表达载体pET32a上，经过EcoRⅠ和SacⅠ双酶切鉴定和序列分析后，得到阳性重组质粒pET32a-σC；将阳性重组质粒pET32a-σC转化到大肠杆菌BL-21感受态细胞中进行诱导表达，经SDS-PAGE和Western-blbtting检测分析，融合表达的蛋白能够与番鸭呼肠孤病毒感染的康复鸭血清发生特异性反应；将融合表达的蛋白纯化后作为包被抗原，建立了检测鸭血清中呼肠孤病毒抗体的间接酶联免疫吸附试验检测方法，此方法中抗原的最佳包被浓度为5μg／ml、标准阳性血清的最适稀释倍数为1：40倍，用此方法对50份鸭血清样品进行检测，并与琼脂糖凝胶扩散试验检测抗体的法相比较，证明此ELISA方法具有良好的特异性和敏感性，本研究为今后鸭呼肠孤病毒诊断试剂盒的研制奠定了基础。

The thymidine kinas gene was cloned from human herpes simplex virus Ⅱ by PCR techniques; eukaryon expressing vector pcDNA3/tk and pcDNA3/angio/tk fusion gene was constructed by DNA recombination; The treating effects of human primary liver cancer strain SMMC-7721 in vitro and nude mouse model transplanted with human liver cancer in vivo are studied.

通过PCR技术，由人单纯疱疹病毒DNA克隆获得单纯疱疹病毒Ⅱ型胸苷激酶基因；通过基因工程技术，构建真核表达质粒pcDNA3/tk及融合基因真核表达质粒pcDNA3/angio/tk；研究其对人原发性肝癌SMMC-7721细胞株的体外杀伤作用及对人原发性肝癌裸小鼠移植瘤模型的治疗作用。

The carboxyl-terminal fragment of approximately 15ku of zonula occludens toxin gene encoding the 264-399 amino acid residue was amplified by PCR and then cloned into the prokaryotic expression plasmids PBCX.

用PCR扩增了MBP-ZOT质粒上的ZOT基因，该基因编码264～399位氨基酸残基C末端的15ku ΔG片段，并与经改造后的质粒PBCX连接。

The stability of exogenous plasmids introduced in BMB171 was correlated with the patterns of replicons, and sizes of plasmids.

用pHT3101、pBMB3305、pBMB1736、pBMB671、pBTL-1和pHV1249等6种外源质粒电转化无质粒突变株BMB171的转化频率，分别是Bt-4Q7、Bt-4D10和Bti。

The 329 bp terminal inverted repeat sequence can't form the conserved fold-back secondary structure as that of many Streptomyces linear replicons. Lacking of typical Streptomyces tap/tpg locus for te-lomere replication, pPR2.3c encodes a protein with two domains resembling the telomere associated protein of Strepto-myces and helicase of Haemophilus respectively. No typical Streptomyces iteron-rep locus for replication from the cen-trally located origin, two DNA fragments containing almost all pPR2 were cloned and introduced by transformation into S.

其端粒末端反向重复序列的长度为329 bp，不能像多数链霉菌的线型质粒那样能形成保守的&折返&的二级结构。pPR2虽然没有参与链霉菌端粒复制的保守的tap/tpg基因，但是pPR2.3c基因编码了一个双结构域蛋白，分别同链霉菌的端粒复制相关蛋白Tap和嗜血杆菌的解旋酶具有相似性。pPR2缺少典型的链霉菌重复序列-复制基因区段，将几乎覆盖全长pPR2的两段DNA进行克隆后，不能转化变铅青链霉菌。

MAIN OUTCOME MEASURES: Identification of the recombinant plasmids and the expressing mRNA and protein in 293-T cells.

主要观察指标：质粒的酶切鉴定及重组质粒在293-T细胞中mRNA及蛋白水平的表达。

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！