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In this study, the DNA fragments coding for amino acids 133～158 of VP1and 20～34 of VP4protein of type Asial FMDV were chemically synthesized and ligated into a tandem repeat 133～158-20～34-133～158. The sequences of signal peptide of Igκ chain and Kozak were fused to the 5'end of this tandem sequence and synthetic oligodeoxynucleotide containing CpG-ISS was fused to downstream of terminal coden of this tandem sequence. And then this long fragment was cloned into the eukaryotic expression plasmid pHook-2, forming a new secreted expression plasmid, named pAS1-E.

在第二章证明了亚洲Ⅰ型口蹄疫病毒VP1蛋白中133～158位氨基酸残基确是一重要B细胞中和抗原表位的基础上，依据第一章获得的口蹄疫病毒亚洲Ⅰ型VP1 cDNA序列及已报道Asial VP4序列，采用真核偏爱密码子化学合成了VP1中编码133～158位氨基酸及VP4中编码20～34位氨基酸这两个抗原表位基因，将其组成133～158-20～34-133～158串联结构，在其5′端加上鼠Igκ链信号肽序列，在翻译调节区加上增强表达的Kozak序列，同时在133～158-20～34-133～158串联结构3'端终止密码子下游加上CpG免疫刺激序列，将这些片段连接后，克隆到真核表达载体pHook-2上，构建成功了DNA疫苗重组表达载体pAS1-E。

Methods The total RNA was acquired from young rabbit particular cartilage, the BMP7 gene was inserted into pcDNA-3.1 to construct eukaryotic expression vector plasmid of pcDNA3.1-BMP7; the target gene-BMP7 was identified respectively by PCR analysis, restriction enounces analysis and nucleotide sequencing; the plasmid was transfected into adult rabbit knee articular chondrocytes, then the expression of BMP7 was detected by in situ hybridization, PCR and Western blotting.

从体外培养的幼兔膝关节软骨细胞中提取总RNA；按GenBank BMP7基因序列化学合成2条引物，采用RT-PCR方法得到BMP7基因；将BMP7基因片段插入到真核表达载体pcDNA 3.1中，构建pcDNA3.1-BMP7真核表达载体质粒；利用双酶切、PCR及核苷酸序列分析鉴定BMP7目的基因；将重组pcDNA3.1-BMP7真核表达载体质粒转染家兔关节软骨细胞，分别采用原位杂交、PCR、Western blotting法测定BMP-7表达。

By means of experim ents of marker transfer,acridine orange sensitivity test,F' curring and mini-chr omosome transformation it is concluded that the F' plasmid is always in an integ rated state in the SIn strains and that the initiation of the replication of the bacterial chromosome is carried on by the integrated F' plasmid.

标记转移、吖啶橙敏感性、F′质粒消除和mini-染色体质粒转化等实验说明，Sin菌株中F′质粒始终处于整合状态，并且在40℃中细菌染色体的复制由整合状态的F′质粒所带动。

The optimal conditions for electroporation transformation of Pichia Pastoris were obtained with the maximum transformation efficiency of 36 transformants/μg plasmid DNA when the intermediate logarithmic phase yeast cells were used for competent cells, and the electroporations were under the conditions of voltage of 1.5kV and 0.15mg / mL plasmid DNA and 0.2 cm cuvettes.

结果表明：当采用对数生长中期的菌体制备感受态细胞、电压为 1.5kV、质粒浓度为 0.15μg/μL和 0.2 cm 电转化杯时，转化效率达到最大值，为每微克质粒 DNA 36个转化子。

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 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。

RT-PCR was a sensitive and stable method. It would be useful for large-scale diagnosis.A length cDNA of CEVd had been cloned into plasmid vector. Biotinylated probes were prepared from these plasmid DNAs. CEVd were examined from the positive and netative materials by Dot-blotting hybridization and imprint-hybridization. The result was exposed to x-ray film. Singles were detected for positive materials spots probed with probe, indicating that under the reaction conditions the probe hybridized with the target viroid specifically.

核酸杂交法检测：利用已经克隆的美国柑桔裂皮类病毒cDNA序列，制备生物素标记的核苷酸探针，采用斑点杂交的方法和组织印迹的方法分别对阳性材料进行杂交检测，通过胶片曝光显示杂交信号，发现在两种方法的检测结果中阳性材料都有强的反应，而阴性材料无反应或较弱，该方法安全可靠、快速准确，适合大量样品的检测。

According to these factors, three sections of experiments have been carried out to determine the plasmid uptakes in mice muscles, using the available and reformed plasmid vectors.

当前，导致裸DNA肌肉注射方法表达效率低的根本问题在于：对注射后DNA是如何进入细胞并得到表达的机制还不清楚。

In this study, the replicon of the largestdetected plasmid pBMB 165 and the smallest plasmid pBMB 175 of strain YBT-1765 werecharacterized.

该菌株至少含有三个内生质粒，本文对其可检测到的最大质粒pBMB165的复制区和最小质粒pBMB175的研究结果如下： 1。

The PTZ plasmid vector was changed to plasmid vector pSP72, which contains both T7 and Sp6 promotors for transcription of antisense (T7) and sense (Sp6) RNA probe in vitro. The results revealed the label rate of cRNA probe is high, the hybrids between RNA-RNA are stable, so the background of hybridization is satisfactory.

本实验将质粒载体PTZ转换为带有T7和Sp6两个启动基因的pSP72载体，故可在体外转录cRNA时同时得到意义（Sp6为启动基因）和反意义（T7为启动基因）探针，实验结果表明，cRNA探针标记率高，杂合物稳定，故所获杂交反应背景较为满意。

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！