质粒

In this experiment, we screen the major protective antigen gene-SOD gene of M. paratuberculosisin order to study the sensitive, specific diagnostic reagent and prophylaxis preparation, especially theDNA vaccine. The SOD gene was amplified from Mycobacterium paratuberculosis C-2 chromosomalDNA by using the PCR technique and cloned into pMD18-T Vector System. We gained a SOD gene of624bp.The recombinant clone was identified byα-complementarity, enzyme digestion and PCRidentification. The result indicated that the recombinant plasmid pMD18-T-SOD was successfullyconstructed. Moreover, through sequential determination and DNASTAR analysis between the clonedSOD gene of M. paratuberculosis C-2 and that of the M.paratuberculosis K-10 strain, the sequentialhomogeneity reached 99%, and the amino acid homogeneity reached 99.5%. The preceding analysisindicated that the SOD gene was very conservative in M. paratuberculosis.

为了研制副结核病敏感、特异的诊断试剂和新型、高效的预防制剂，尤其是DNA疫苗，本研究筛选了M.paratuberculosis主要保护性抗原SOD基因，以M.paratuberculosis C-2染色体DNA为模板，以SOD基因的特异性引物进行PCR扩增，获得了624bp的SOD基因，通过T-A克隆技术，将PCR产物克隆至pMD18-T Vector中，以质粒大小、酶切分析、PCR扩增及序列分析鉴定重组克隆，成功地构建出克隆质粒pMD18-T-SOD，序列测定及DNASTAR分析表明，所获得的M.paratuberculosis C-2 SOD基因与Gen Bank中M.paratuberculosis K-10 SOD基因的大小完全一致，两者核苷酸序列的同源性为99％，氨基酸序列的同源性为99.5％，表明该基因在副结核分枝杆菌中是高度保守的。

It also can be used as alternative antigen of newgeneration vaccine.In this experiment,we screen the major protective antigen hsp65 gene of MAP in order to developnew vaccine especially the DNA vaccine for the prevention of paratuberculosis disease.The hsp65 genewas amplified from MAP C-2 chromosomal DNA by using the PCR technique.We gained a hsp65 gene of 1 626bp.Then PCR product was cloned into pGEM-T vector by T-A clone technique and therecombinant clone was identified by plasmid size,enzyme digestion and PCR identification.The cloneplasmid of pGEM-T- hsp65 was successfully constructed.The nucleotide sequence and deduced aminoacid sequence ofclone gene was analyzed by DNASTAR software.The result indicated that the size ofhsp65 gene consist with M.paratuberculosis K-10 strain in GenBank and the sequential homogeneityreached 99.1%,the amino acid homogeneity reached 99.3%.The preceding analysis indicated that thehsp65 gene was very conservative in M.paratuberculosis.

为了研发预防副结核病的新型疫苗尤其是DNA疫苗及相关蛋白功能，本研究选择了MAP的主要保护性抗原Hsp65蛋白，以副结核分枝杆菌C-2株染色体DNA为模板，以hsp65基因的特异性引物进行PCR扩增，获得了1 626bp的hsp65基因，通过T-A克隆技术，将PCR产物克隆至pGEM-T Vector中，以质粒大小、酶切分析、PCR扩增及序列分析鉴定重组克隆，成功地构建出克隆质粒pGEM-T-hsp65，以DNASTAR软件分析了所克隆基因的核苷酸序列和推导的氨基酸序列，结果表明，所获得的hsp65基因与GenBank中MAPK-10株该基因核苷酸大小完全一致，两者核苷酸序列的同源性为99.1％，氨基酸序列的同源性为99.3％，表明该基因在副结核分枝杆菌中高度保守。

The papA gene was amplified from the pilus adhesive cluster of UPEC132, and then was cloned into plasmid pUC18. The recombinant plasmid was identified and subjected to DNA sequencing.

采用此引物扩增132菌株p菌毛粘附基因群的papA基因，扩增产物克隆入质粒，选取阳性重组质粒进行核苷酸序列分析。

In order to study the expression and functional characteristics of dehydrin gene during different growth, and make the polycolonal antibody, using wheat plumelet under the water deficit condition as experiment material, and its total RNA was extracted. The target dehydrin gene through RT-PCR was got, connect to the cloning vector PUCM-T, recombination expression plasmid PET-32a-wzy1-1 was constructed according to recombination of cloned vector PUCM-T-WZY1-1 of wheat dehydrin gene in this experiment.

为研究小麦在不同时期脱水素基因的表达情况和生物学功能及抗体制备，以小麦幼芽为材料，经干旱胁迫处理后，提取总RNA，通过RT-PCR得到小麦类脱水素基因片段(WZY1-1)，再连接至克隆载体PUCM-T，并成功构建重组表达质粒PET-32a-wzy1-1，将阳性重组质粒转化于受体菌BL21(DE3)感受态细胞中，经IPTG诱导表达，进行表达产物的聚丙烯酰胺凝胶电泳检测。

The synthesized cDNA is a continuous band less than 6. 0kb shown on x-film through α〓PdCTP which was inserted in the first strain of cDNA. The titer of the Ha-cDNA library was about 1. 5×10〓cfu/ml which could represent all protein-encoding sequences from Hz-AM1 cells; Namely, the cDNA library has representativity. After the successful construction of the Ha-cDNA library, the open-reading frame of vp39 gene of Heliothis armigera was then fused in frame with the Ga14-DNA-binding domain in the pGBT9 plasmid and the fusion protein Ga14-VP39 was expressed in yeast to be used as bait protein.

在成功构建棉铃虫细胞Hz-AM1cDNA文库的基础上，本文接着将棉铃虫核型多角体病毒衣壳蛋白VP39（HaNPV-VP39）基因克隆到酵母双杂交系统(yeasttwo-hybrid system)中的质粒pGBT9上。pGBT9上含有酵母菌转录激活因子GAL4 DNA结合域的编码区（GAL4-DBD），使克隆到质粒pGBT9上的HaNPV-vp39基因与GAL4-DBD基因融合，在酵母菌中表达DBD-vp39融合蛋白，该融合蛋白即为酵母双杂交系统中的诱饵蛋白。

At the same time, intact shuttle-plasmid pZ189 was also introduced into the host cell which was used for detection of nontargeted mutation frequency. After replicating in cells, progeny plasmid DNA was isolated and transformed into E coli. MBM7070.In the presence of ampicillin, X-gal and IPTG, pZ189-transformed E. Coli MBM7070 forms blue colony if the plasmid contains an active SupF suppresser tRNA gene, and white or light blue colony if the SupF tRNA gene is inactive.

但是由于检测非定标性突变需同时引入穿梭质粒pZ189在细胞中复制，并回收质粒DNA，通过转化宿主菌E.coliMBM7070，在含x-gal和IPTG的氨苄指示板上培养，如果用于检测突变的靶基因SupF tRNA基因发生突变，则菌落色泽为白色或淡蓝色，而正常者为兰色。

Result find that four cell lines express stably miRNA, and miRNA regulate expression of targe gene in stable cells.

软琼脂集落实验证实靶向hTERT的质粒和同时靶向两个基因的质粒都能够抑制CNE-2细胞的集落形成能力。

In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5′ end sequence was truncated by primer, then the obtained truncated MGF des(1-3MGF cDNA (321 bp) was subcloned in pET32a vector to construct a prokaryotic recombination expression plasmid.

通过RT-PCR从拉伸刺激的人成骨细胞中克隆MGF cDNA序列，并去除5'端9 bp的序列，使N端缺少对肠激酶(Enterokinase， EK)具有抑制作用的脯氨酸，将截短型MGF des(1-3 MGF cDNA序列克隆入pET32a质粒，构建重组表达质粒。

Methods The HPV16E7 gene was cut to three parts, amplified by PCR, and connected individually with BPVL1 on plasmid PUC. With PVL1393 as a transfect vector, the BPVL1/HPV16E7 recombinants were transfected to baculovirus which subsequently expressed the chimeric BPVL1/HPV16E7 protein in the SF－9 cells. The recombinant proteins were then purified by centrifuge, sonication, sucrose and CsCl ultracentrifuge, and were identified by SDS-PAGE, Western blot, ECL and TEM.

HPV16E7基因分3段经PCR扩增后分别克隆入连有BPVL1的质粒PUC形成BPVL1-HPV16E7；以质粒PVL1393为载体将BPVL1-HPV16E7基因转染杆状病毒并在昆虫细胞中进行表达；用超声粉碎和蔗糖超离、氯化铯梯度离心等方法纯化以及免疫印迹、ECL、透射电镜等方法鉴定表达产物。

In order to prevent linear PCV2 cyclization, PCR mutagenesis was used to construct the first molecular clone (pSK-2PCV2) by ligating two copies of the complete PCV2 genome with the pBluescript SK vector. In addition, pSK-PCV2 and ds-PCV2 were constructed. PK-15 cells were transfected with above three infectious clones.

本研究应用PCR诱变技术解决了外源片段易于自连的难题，成功将2个PCV2 SD1株全基因组(DQ346683)头尾相接插入到真核生物表达载体pSK的多克隆位点中，构建重组质粒pSK-2PCV2；另外课题组成功构建含单个PCV2全基因组的pSK-PCV2和自身环化质粒ds-PCV2。

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！