质粒体

Orange-spotted grouper is a benthic protogynous marine fish with sex inversion. It is one of the most important culturing fish in the southeast of Asia. As in other vertebrates, its growth is mainly controlled by growth hormone . Few data are available concerning the regulation of GH in grouper. In this paper, the regulation of growth hormone release from pituitary of orange-spotted grouper and its mRNA level was determined by radioimmunoassay and RNase protection assay.

中文题名斜带石斑鱼生长激素分泌及其基因表达调控的研究副题名外文题名 Regulation of growth hormone and its gene expression in orange-spotted grouper，Epinephelus coioides 论文作者冉雪琴导师林浩然学科专业水生生物学研究领域\研究方向学位级别博士学位授予单位中山大学学位授予日期2003 论文页码总数140页关键词生长激素基因表达石斑鱼馆藏号BSLW /2003 /Q959 /39 应用分子克隆技术，构建了斜带石斑鱼生长激素(Growth hormone，GH)基因重组质粒，在大肠杆菌细胞中获得高效表达，亲和层析纯化斜带石斑鱼GH融合蛋白做为抗原，免疫家兔制备特异性抗体，通过酶联免疫实验、放射免疫测定、离体脑垂体静态孵育和液相杂交／核糖核酸酶保护试验及化学发光检测技术，研究了斜带石斑鱼GH分泌及其mRNA表达的调控机制，对于斜带石斑鱼的生长、繁殖、人工养殖等具有重要意义。

The DNA of Yak PrP expressed plasmid extracted from the recombinant expressed bacterium was identified by PCR amplification and two-ezyme digestion. The expressed product was obtained from the recombinant bacterium inducted under the optimized expressed condition (37℃, 1 mmol/L IPTG, 6 h), and it was determined by SDS-PAGE and Western blotting. The GST-BoPrP(23—242) fusion proteins were collected by two ways: the first is to purify and renature from the inclusion bodies; the second is to separate by SDS-PAGE.

将保存的含有牦牛PrP基因重组表达菌提取质粒DNA，进行PCR扩增和双酶切鉴定，并在优化诱导表达条件(37℃，1mmol/L IPTG，6h)下，获得的表达产物进行SDS-PAGE和Western blotting检测；将鉴定之后的重组表达菌诱导表达后，通过两种方法回收GST-BoPrP(23～242)融合蛋白：其一，是从包涵体中提取和复性GST-BoPrP(23～242)融合蛋白；其二，是通过SDS-PAGE直接分离并纯化GST-BoPrP(23～242)融合蛋白。

Methods: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B.

用DNA重组技术，将溶酶体的靶向信号肽KFERQ连接在RTA的羧基端；将构建好的重组质粒pKK223.3-RTA和pKK223.3-RTA-KFERQ转化感受态大肠杆菌JM109，经IPTG诱导表达RTA和RTA-KFERQ蛋白；重组蛋白质用Blue-Sepharose6B亲和柱纯化，以MTT法分别测定纯化后的RTA与RTA-KFERQ蛋白对体外培养的HEPG2、Hela、A5493种细胞的毒性作用。

Methods: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B.

用DNA重组技术，將溶酶体的靶向信號肽KFERQ连接在RTA的羧基端；將构建好的重组质粒pKK223.3-RTA和pKK223.3-RTA-KFERQ转化感受態大肠桿菌JM109，经IPTG诱导表达RTA和RTA-KFERQ蛋白；重组蛋白质用Blue-Sepharose6B亲和柱纯化，以MTT法分别测定纯化后的RTA与RTA-KFERQ蛋白对体外培养的HEPG2、Hela、A5493种细胞的毒性作用。

During the past 3 years, 2 independent experiments of sperm-mediated gene transfer in domestic silkworms were carried out respectively in 2000 and 2002 with the method direct injection of copulation thecae.

在同源重组绿色荧光蛋白表达载体pG350的基础上，以pMD18-T为质粒载体，用egfp基因替换gfp基因，构建了家蚕核型多角体病毒早期即刻蛋白基因启动子BmNPV-IE控制下的增强型绿色荧光蛋白表达载体pMD-Fib-IE-EGFP。

The subcellular localization of TIAR full length and truncated mutants were observed by using fluorescent microscopy.

酶切鉴定及测序结果显示TIAR全长及缺失体的pEGFP-C3重组表达质粒构建成功。

An in vitro transcription recombinant plasmid 5T1d was obtained after subcloning the target gene of clone 5T1d,which contained mutant HCV 5 non-coding region cDNA,into a transcriptive vector pCDNA Ⅰ.

采用含HCV5非编码区(5NCR)缺失突变的重组质粒5T1d，将其目的基因亚克隆到体外转录载体pCDNAⅠ多克隆位点上，获得转录重组体5T1d。

Methods The recombinant plasmid of CPB, pET-21a-CPB, was constructed and transformed into E. coli BL21(DE3). The expression was induced in 25℃and 37℃, respectively. The obtained inclusion bodies were dissolved in different denaturing solution systems. The protein concentration was determined in order to indicate the solubility of CPB inclusion bodies in the denaturing solution. And probable mechanism for the solubility differences was illuminated by the analysis of unreduced SDS-PAGE.

方法构建CPB重组质粒pET-21a-CPB，将其导入表达菌株BL21（DE3）中，分别在25 ℃和37℃进行诱导表达；所得包涵体分别用不同浓度尿素添加不同还原剂溶解，以溶液中的蛋白质浓度判定其溶解性，利用非还原SDS-PAGE分析其溶解性提高的原因。

DNA sequencing confirmed that the constructs did have Chlamydial encoding genes in it which were correct in translation frame and sequence.

DNA 序列分析证实重组质粒含有相应的衣原体基因，其读码框架正确。

Results The eukaryon expression system pcDNA3.1/myc-HisC-bFGF could express the target protein bFGF in vitro. The recombinant plasmids, pcDNA3.1/myc-HisC-bFGF and pCD2-VEGF121 were transferred into muscles flap in vivo successfully. The active proteins bFGF and VEGF121were expressed at high levels.

结果 构建的pcDNA3.1/myc-HisC-bFGF真核表达体系成功转染体外培养HeLa细胞，目的基因在mRNA水平和蛋白水平均有表达。pcDNA3.1/myc-HisC-bFGF和pCD2-VEGF121重组质粒分别转染在体肌瓣，获得外源基因高水平表达。

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！