克隆

Results After screening, 59 subtracted library clones were isolated which were specific for strain VIB72, and the DNA sequences of these clones were determined. Seventeen fragments showed high homology to the genes of known functions in other bacteria. This includes soluble lytic murein transglycosylase, mobilization protein, transposase (IS66), resistance-related protein (metallo-beta-lactamase and acetyltransferase family), toxin protein (DT-201 and alveicin A immunity protein), ATP-dependent endonuclease of OLD family like protein, SocE and GTP-binding protein HflX (high frequency of lysogenization).

通过对差减文库筛选，分离到59个对菌株VIB72的克隆，并对这些克隆的DNA序列进行了测定。17个基因片断与其它细菌的已知功能的基因有较高的同源性，其中包括可溶性溶胞壁质转糖基酶、转移蛋白MobA和MobC、转座子IS66、抑制相关蛋白（金属β-内酰胺酶和乙酰转移酶家族）、毒素蛋白（DT-201和alveicin A免疫蛋白）、与OLD 家族相似的ATP依赖性核酸内切酶以及SocE 和GTP结合蛋白HflX。

Results of sequence and homology analysis show that MD 1 has 97% similarity with mark in maize chloroplast genome,a gene encoding RNA maturase involved in group Ⅱ intron splicing of RNA transcript;MD2 has 99% similarity with the gene serine/threonine phosphorylase 2C in Sporobolus stapfianus;and MD3 has 99% similarity with rice the gene encoding metacaspase,an arginine/lysine-specific cysteine protease.

序列分析和同源性比对表明，MD1与编码成熟酶的玉米叶绿体基因matK有97%的相似性，MD2与极端耐旱植物Sporobolus stapfianus编码丝氨酸/苏氨酸蛋白磷酸酶的PP2C基因有99%的相似性，MD3与属精氨酸/赖氨酸特异性半胱氨酸蛋白酶类的水稻metacaspase酶基因有99%的相似性。根据MD2片段序列，结合电子克隆和RT-PCR方法，克隆出一条1731 bp的全长cDNA序列，它编码388个氨基酸。

A pair of primers were designed and synthesized based on the nucleotide sequence of coat protein gene of TAV from England.

将其克隆到 pGEM Teasy中，经酶切和序列分析表明所克隆的是TAVCP基因，与已知的 6个株系相应序列同源性均在 96 。4%以上

Methods: total rna was isolated from hepatocellular carcinoma cell line hepg2. subsequently, a 1kb fragment of hccr 2 encoding region was amplified by rt pcr and cloned into promega teasy vector. after confirmed by dna sequencing, the full length encoding region of hccr 2 was amplified by pcr and cloned into pires2 egfp. the recombinant eukaryotic expression vector was confimed by dna sequencing.

从人肝癌细胞株hepg2提取rna，利用rt pcr方法，先克隆出一包括hccr 2编码区的长约1 kb的片段，并将其与t载体连接并转化，测序证实无误后，以hccr 2/t载体为模板，再通过pcr克隆出hccr 2的编码区序列，将其连接到pires2 egfp真核表达载体，并通过测序获得证实。

METHODS: Total RNA was isolated from hepatocellular carcinoma cell line HepG2. Subsequently, a 1kb fragment of HCCR2 encoding region was amplified by RTPCR and cloned into Promega TEasy vector. After confirmed by DNA sequencing, the fulllength encoding region of HCCR2 was amplified by PCR and cloned into pIRES2EGFP. The recombinant eukaryotic expression vector was confimed by DNA sequencing.

从人肝癌细胞株HepG2提取RNA，利用RTPCR方法，先克隆出一包括HCCR2编码区的长约1 kb的片段，并将其与T载体连接并转化，测序证实无误后，以HCCR2/T载体为模板，再通过PCR克隆出HCCR2的编码区序列，将其连接到pIRES2EGFP真核表达载体，并通过测序获得证实。

The efficiency of selection was monitored by comparing the number of phage recovered from the acid elution and cell lysate in each round,and by testing EGFR binding specificity of polyclonal phagescFv on CHOEGFRGFP1 and CHOK1 cell with cell ELISA. Bacterial PCR was used to select clones containing a 1 kb insert. Cell ELISA was used to determine EGFR binding specificity of monoclonal pscFv on EGFR positive and negative cell. The number of individual EGFRbinding clones was determined with nucleotide sequencing. Results 500fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection.

以稳定转染的CHOEGFRGFP1细胞和未转染的CHOK1细胞分别作为EGFR阳性和阴性细胞，采用负筛选的方法进行筛选；通过比较每轮投入及洗脱出噬菌体的效价比以及细胞ELISA检测多克隆pscFv与阳性、阴性细胞结合情况对筛选过程进行监测；采用菌落PCR挑选含有全长scFv片断的菌落，进一步用细胞ELISA检测单克隆pscFv与EGFR阳性及阴性细胞结合特异性；挑选EGFR特异性单克隆pscFv采用DNA测序法确定克隆多样性。

Results 500fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection. A signal significantly greater than background binding was observed from the third to the fifth round of selection on CHOEGFRGFP1 and CHOK1 cell, but a part of polyclonal scFv bound EGFR specially. About 45% of the selected clones contained a fullsized insert of 1 kb. One unique human antiEGFR scFv (F4scFv) was isolated by analyzing with cell ELISA and DNA sequencing.

结果 经过5轮筛选，细胞裂解液中洗脱出噬菌体效价有500倍以上增长；细胞ELISA结果显示多克隆pscFv与CHOEGFRGFP1细胞和CHOK1细胞均有明显结合，但有部分特异性结合EGFR；菌落PCR显示约45％克隆中含有完整的1kb scFv片断；经细胞ELISA、 DNA测序检测共获得1株EGFR特异性单链抗体，命名为F4scFv。

Cell ELISA was used to determine EGFR binding specificity of monoclonal pscFv on EGFR positive and negative cell. The number of individual EGFRbinding clones was determined with nucleotide sequencing. Results 500fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection.

以稳定转染的CHOEGFRGFP1细胞和未转染的CHOK1细胞分别作为EGFR阳性和阴性细胞，采用负筛选的方法进行筛选；通过比较每轮投入及洗脱出噬菌体的效价比以及细胞ELISA检测多克隆pscFv与阳性、阴性细胞结合情况对筛选过程进行监测；采用菌落PCR挑选含有全长scFv片断的菌落，进一步用细胞ELISA检测单克隆pscFv与EGFR阳性及阴性细胞结合特异性；挑选EGFR特异性单克隆pscFv采用DNA测序法确定克隆多样性。

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。

In the present paper, the Sox gene expression analysis of different tissues from the Trionyx sinensis was studied by using RTPCR, and Sox gene fragments of expression from the testicle, brain and spleen were cloned using RTPCR products. The results show that Sox gene has specific expression in the testicle, brain, spleen, cardiac muscle and kindney and hasn't expression in muscle, liver and ovary. The results of clone reveal that the Sox genes of expression in the testicle are TSSox1 and TSSox 4, and those are TSSox2 and TSSox4 in the brain, and that is TSSox4 in the spleen. This suggests that the Sox gene act important role not only on the sex determination, but also on the development of neural system, immunocyte system and the differentiattion of male germ cell.

本文采用RT—PCR技术，研究了中华鳖不同组织Sox基因的表达，并通过PCR直接克隆法，分析了来自睾丸、脑和脾组织中的Sox基因序列结果表明在中华鳖的成体组织中，Sox基因在脑、心肌、肾、脾和雄性的睾丸组织中均有不同程度的表达，而在肌肉、肝脏和雌性的卵巢中则无表达，显示该基因具有组织表达特异性克隆分析显示，在睾丸组织中表达的是TSSox1和TSSox4基因，而在脑组织中表达的是TSSox2和TSSox4基因，脾组织中表达的是TSSox4基因此结果表明Sox基因不仅在性别决定中起作用，还可能在神经系统、免疫系统多种组织中起重要作用

Lugalbanda was a god and shepherd king of Uruk where he was worshipped for over a thousand years.

Lugalbanda 是神和被崇拜了一千年多 Uruk古埃及喜克索王朝国王。

I am coming just now,' and went on perfuming himself with Hunut, then he came and sat.

我来只是现在，'歼灭战perfuming自己与胡努特，那麼，他来到和SAT 。