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The plasmid of pMD-T-GO and pPIC3.5K were extracted from JM109 strain and then glycolate oxidase gene fragment was cloned into the expression vector (pPIC3.5K). The recombinant plasmids were transformed into E.coli TOP 10Fˊ. The recombinant clones were identified by PCR, digested with SnaB I /Not I and then sequenced.

本文首先从大肠杆菌JM109中提取质粒pMD-T-GO和pPIC3.5K，将菠菜乙醇酸氧化酶基因c DNA片段克隆至表达载体pPIC3.5K，转化进E.coli TOP 10Fˊ，利用设计的引物进行PCR扩增、SnaB I和Not I酶切鉴定，最后进行序列分析。

The different fishes which are employed in the present studies are wild-type salmon, cultured salmon of freshwater and seawater, sea perch and fat greenling. The complete CT gene sequences of salmons are obtained by PCR amplification. The partial CT sequences of sea perch and fat greenling are obtained by in vitro cloning PCR method. Alignment of obtained CT sequences with other fish CT shows that CT appears to be well conserved among the same family. And the relative in taxonomy is far away, the similarity of different fish CTs is low. On the contrary, the closer the relative in taxonomy is, the higher the similarity of different fish CTs is. The sCT is expressed in pGEX-4T-X by recombinant form . We also succeed in the research of sCT expression alone by expression PCR. In addition, sCT antiserum is obtained using GST-sCT as antigen, and the high titer is tested by double immunodiffusion. In the rat bioassay, administration of 50 μg recombinant protein evoked significant hypocalcemia at 1 h after the data are analyzed by t-test.

本文用PCR方法克隆了野生鲑鱼、养殖鲑鱼的降钙素基因，并应用体外克隆PCR的方法首次克隆出鲈鱼、六线鱼降钙素的部分基因序列，通过对克隆的降钙素序列的比较研究，结果显示同一科的鱼降钙素序列保守性较高，同时，根据降钙素的部分氨基酸序列进行了降钙素相似性的比较研究，结果显示在分类学上，分类地位较远的鱼，其降钙素相似性较低，分类地位越接近的鱼，其降钙素相似性越高；我们利用谷胱甘肽S-转移酶(Glutathions S-transferase，GST)融合表达载体pGEX-4T-X对克隆的鲑鱼降钙素基因进行了融合表达研究，应用表达PCR的方法对降钙素基因的独立表达进行了初步的研究探索，并将纯化的融合蛋白作为抗原，获得了高效价的兔抗鲑鱼降钙素免疫血清；生物活性研究表明，大肠杆菌表达的融合蛋白具有显著的降血钙作用。

For different methods (moment solution and maximum likelihood estimation) and different models (homoscedastic and heteroscedastic models),more signifiant correlation was found between LOD scores than between the estimators of recombinant fraction ,Maximum likekihood method under homoscedastic model was then used to estimate recombinant fractions between quantitative trait lci controlling yield componments and 51 restriction fragment length polymorphism makers on 11 chromosomes of rice Oryza sativa L.

本研究根据对估计标记-数量性状基因座位之间重组率的两种分析方法、两种方差模型(QTL 基因型之间的方差同质和异质模型)的分析，揭示了LOD值在标记-QTL连锁检测上所得结果的相关性高于重组率估计值的相关性。

Recombinant bovine prion protein was treated by chemical solution, and the treated Recombinant bovine prion protein was intracerebrally inoculated in the brain. The prion gene expression was quantified and the proteinase resistant protein was detected by western-blotting.

对重组牛朊蛋白进行了化学处理后脑内接种金黄地鼠，200天后对朊蛋白抗性进行了western-blotting检测，对脑组织朊蛋白基因的表达进行了分析。

The ORF of profilin of Caryota mitis pollen was amplified by RT-PCR using specific primers and cloned into the expression vector pET-28a. The recombinant Caryota mitis pollen profilin was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by affinity chromatography using Ni(superscript 2+) coupled sepharose.

然后设计带有酶切位点的特异性引物，采用RT-PCR获得整个短穗鱼尾葵花粉profilin的开放阅读框，将其与pET28a载体连接并转化大肠杆菌BL21(DE3)进行诱导表达，通过Ni(上标 2+)亲和层析柱对重组蛋白进行纯化，采用Western-blot检测其IgE结合活性。

Methods BALB/c mice were inoculated muscularly with HIV-2 gag recombinant DNA vaccine, or recombinant fowlpox virus, or both.

大量制备并纯化HIV-2 gag重组DNA疫苗和重组鸡痘病毒，以肌肉注射的方式免疫BALB／c小鼠，ELISA法检测小鼠血清HIV-2抗体，流式细胞仪测定CD4^＋、CD8^＋T淋巴细胞亚类数量，乳酸脱氢酶释放法检测脾CTL对HIV-2靶细胞的杀伤活性。

Methods The recombinant sequence for two CEA epitopes was tandemly engaged and inserted into-upstream of in-frame-varied HSP70 of Mycobacterium tuberculosis to construct a gene vaccine.Balb/c mice were muscularly injected with recombinant DNA vaccine;negative control (mice were injected with normal saline), positive control (mice were injected with DNA vaccine harboring tandem CEA epitopes plus aluminium hydroxide adjuvant) and experimental group(mice were injected with PCITri CEA625-667-met HSP70) were set up.

在已经构建含有变异热休克蛋白序列的基础上，插入重组CEA串联表位的编码片段获得CEA串联表位-HSP融合基因疫苗。3次肌肉注射免疫Balb/c小鼠，设立注射生理盐水的阴性对照组、注射氢氧化铝佐剂混悬CEA串联表位的阳性对照组及注射pCITriCEA625-667-mtHSP70的实验组。

In our experiment, the GADes gene from rat brain was cloned and expressed in E.coli, so as to make the recombinant GADes produced by gene technology for clinical application and further study on the relation between GAD and type 1 diabetes mellitus.Firstly, with the pUClS/GADes recombinant plasmid as template, the full-length encoding sequence of GADes was amplified by PCR, and cloned into pGEM-T vector. Secondly, the target gene was cut by EcoRI and inserted into restriction sites of pET42a vector for expression.

本研究首先用PCR方法，以pUC18/GAD_(65)为模板，扩增目的基因，克隆到pGEM-T载体中，构建pGEM-T/GAD_(65)重组质粒，然后用EcoRI酶切，切下的目的基因片段插入pET42a融合型表达载体中，经酶切鉴定和测序，证实插入方向、读码框架正确；重组表达质粒转化大肠杆菌BL21(DE_3)中诱导表达，表达产物经亲和层析纯化后获得了融合蛋白GST-GAD_(65)。

Recombinant adenovirus particles were produced after 293 cells transfected with recombinant Ad genomic plasmid DNA digested with PacI, then they were further amplified in 293 cells.

纯化所得腺病毒滴度约为8×1012pfu/L，当MOI=100时，腺病毒感染HepG1细胞的效率＞75％，Western Blot证实在感染重组体腺病毒的细胞中有相应基因产物的表达。

After the recombinant plasmid pAd-VP linearized with PacI transferred into HEK-293A cell,the recombinant virus was isolated and purified in HEK-293A cells by three times plaque purification.

RT-PCR 检测证明目的基因在 mRNA 水平上可有效表达；应用 O 型口蹄疫病毒标准阳性血清进行间接荧光抗体试验，在 rAd-VP 感染的 HEK-293A 细胞的胞质可见清晰荧光。

Do not gum up the work by telling mother what we are going to do .

别告诉母亲我们要干的事，那会把事情搞糟的。

In recent years, application of the partial prestressed concrete beam s is developed.

近年来，随着部分预应力砼梁应用的推广，发现我国规范所采用的名义拉应力法的计算结果不稳定。