质粒

Wtp53 has obvious antiblastic effect towards Y79 cells, and the apoptosis rate of Y79 cells was the highest after transfection mediated by ultrasound-microbubble.

结论wtp53基因对RB瘤细胞具有较明显的抑制生长的作用；质粒+微泡+超声辐照，质粒+脂质体转染，以及质粒+超声辐照，均可促进Y79细胞的凋亡，但超声微泡介导的转染引起的凋亡率高于脂质体转染，而二者的凋亡率又明显高于质粒+超声转染。

METHODS: Tum5 gene was amplified from plasmid pSPORT1Sfi by PCR technique and subcloned into the expression plasmid of lentiviral vector, pGCFU, to generate the lentiviral expression vector, pGCFUTum5. The correct Tum5 gene was confirmed by endoenzyme digestion and sequencing. Recombinant lentiviruses were produced by 293T cells following the cotransfection of pGCFU Tum5 and packaging plasmids-pHelper1.0and pHelper2.0. The resulting recombinant lentiviruses (GCFUTum5) which carried Tum5 and EGFPgene were then used to infect human umbilical vein endothelial cells.

采用PCR技术从含有tumstatin基因的质粒克隆模板 pSPORT1Sfi钓取Tum5基因，并将基因克隆到慢病毒载体表达质粒pGCFU中，构建慢病毒载体表达质粒pGCFUTum5，通过酶切、测序验证Tum5基因后，将pGCFUTum5质粒和包装质粒pHelper1.0，pHelper2.0共同转染人胚胎肾上皮细胞系293T细胞，获得携带Tum5基因和EGFP基因的重组慢病毒GCFUTum5，并转染靶细胞人脐静脉血管内皮细胞。

Methods The LPL cDNA was isolated from the human epiploon adipose tissue by means of RT-PCR. The LPL cDNA was ligated into the pcDNA3.1Zeo. The recombinant pcDNA3.1Zeo-LPL cDNA was identified by endonucleases, PCR and DNA sequencing. COS-1 cells were transfected with the recombinant LPL gene plasmid using Lipofectamine 20001M The LPL mass in cells and the culture medium were determined by a Markit-M LPL Kit. Spectrophotometry was used to measure the LPL activity.

采用RT-PCR法从人大网膜脂肪组织获取LPL cDNA基因，以pcDNA3.1Zeo质粒作为载体，运用基因克隆技术构建野生型人LPL cDNA重组质粒，限制性内切酶酶切、PCR以及双向测序鉴定重组质粒；运用Lipofectamine 2000将重组pcDNA3.1 Zeo-LPL cDNA质粒导入COS-1细胞，RT-PCR法检测LPL mRNA，ELISA法和比色法分别检测细胞裂解液及细胞培养基中LPL浓度及活性。

Recombinant plasmid pSVH 7 DNA of avian influenza virus H7 subtype heamagglutinin gene was encapsulated with DC-chol/DOPE liposomes and PC/chol/SA liposomes separately. Two-week old SPF chickens were intramuscularly inoculated with 50 μ g/0.2ml of the liposome entrapped PSVH 7 DNA. Four-weeks later, each chicken was challenged with 0.1ml 〓 AIV . One week after the challenge, the secretion of the cloacas was collected and transfected to chicken embryos to isolate the virus. The virus was isolated from 6/6 of the control group, 1/6 of the naked DNA group, 1/6 of the PC/chol/SA entrapped DNA group and 0/6 of the DC-chol/DOPE liposome entrapped group. The HI antibody titers (log2) of the four groups were 6. 83±0.98, 7. 0±1. 26, 7. 83±1. 17 and 8. 00±0.89 respectively 1-week after challenge, and 8. 5±0.55, 8. 17±0.82, 8. 68±0.45 and 9. 33±0.54 respectively 2-week after challenge. The results showed that inoculation of liposome entrapped DNA significantly enhanced resistance to virosis in animals.

将含禽流感病毒H7亚型血凝素基因的重组质粒pSVH7用DC-chol阳离子脂质体和胆固醇／卵磷脂／十八胺脂质体包裹，免疫2周龄SPF鸡，4周后用同型禽流感病毒进行人工感染，1周后采集泄殖腔分泌物分离病毒，结果未免疫组6／6分离到病毒，裸质粒DNA免疫组1／6分离到病毒胆固醇／卵磷脂／十八胺脂质体包裹DNA免疫组1／6分离到病毒，DC-chol脂质体DNA免疫组没有分离到病毒（0／6）：人工感染后1周各组的HI抗体（Log2）分别为6.38±0.98，7.00±1.26，7.83±1.17，8.00±0.89，2周后为8.50±0.55，8.67±0.82，8.68±0.45，9.33±0.52，脂质体包裹组在同期均高于未免疫组和裸DNA免疫组，表明脂质体包裹质粒DNA免疫动物后，能增加动物对病毒感染的抵抗力和反应能力。

HRP was coupled with the fluorochrome FITC and encapsulated with liposome . pEGFP-N1 plasmid (expressing green fluorescent protein) with encapsulation in commercially available liposome was prepared. The eyes were enucleated 1, 4 and 8 weeks after zonule rupture and anterior segments comprising lenses were incubated in medium containing one of these components. Cryo-sections were made and translocation of fluorescent macromolecule from the medium into the lens and green fluorescent protein expression in the epithelium were observed by fluorescent microscopy.

制备FITC标记的HRP脂质体；制备pEGFP-N1质粒（表达绿色荧光蛋白的质粒），并用脂质体包裹；豚鼠悬韧带部分离断后1周、4周、8周取含晶状体的眼前段标本分别在含有FITC-HRP复合物、pEGFP-N1质粒的培养基中孵育，冰冻切片，荧光显微镜观察FITC-HRP复合物进入晶状体和pEGFP-N1质粒在晶状体内表达的情况，比较悬韧带离断侧与非离断侧的差异。3。

One was the pqsA' positive expression plasmid constructed by cloning the pqsA'-lacZ fusion digested from pYHP441 into miniCTX-1, whose sequence was then integrated into wild type PAO-1 chromosome by biparental mating procedure. The other was pqsA-E operon knock-out plasmid whose sequence between pqsD and pqsE operon was inserted by tetracycline cassette by the site specific insertion mutagenesis strategy and the mutant constructed by biparental mating of S17-1 that harbored the plasmid and pqsA' positive expression mutant.

一种是酶切质粒pYHP441获得pqsA'-lacZ片段后，亚克隆入质粒miniCTX-1中，构建成PqsA'的阳性表达质粒，随后将构建的质粒，通过双亲交配过程整合入野生型铜绿假单胞菌株PAO-1染色体组中；另一种是通过点特异插入诱变策略，将四环素基因盒插入启动子pqsD和pqsE之间，构建的阴性质粒转化入大肠杆菌S17-1株后，和上述pqsA'阳性表达突变株进行双亲交配过程。

TUNEL method was used to detected the apoptosis in the artery. Results:①The MSC cocultured with VSMC expressed smooth muscle a-actin, calponin and CD31, no cells positive for calponin and CD31 were detected in the control group; and a lot of filaments were observed in the co-cultured MSC by electron microscopy.②We gain dual-stable expression of AT2R gene medicated by doxycycline regulatable system of mesenchymal stem cells.

单独培养及VSMC条件培养液培养的MSC仅SM-α-actin表达阳性；(2)Dox-on系统由四个质粒组成：调节质粒pUHD17-1 hyg，荧光报告质粒pUHC 13-3，筛选质粒pSV2neo以及含目的基因AT2R的应答质粒pUHD10-3/AT2R，以上质粒经鉴定均与其背景资料相符合，可用于实现目的基因片段在靶细胞中可调控表达研究；(3)本实验通过连续两个回合的转染及抗生素压力筛选，经Dox诱导表达后获取出低背景、高诱导表达AT2R基因的克隆。

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length P1, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length P1, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3. 1 and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus , which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3. 1/IFN which includes the gene IFN-α of cattle. Subsequently, Recombinant plasmids were injected to cattles with or without pcDNA3. 1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies titers were analyzed by micro-neutralization assay.

为研制带有O型口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)China99株结构蛋白基因及多个非结构蛋白基因的DNA疫苗，本研究通过定点突变方法和PCR扩增方法，获得包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C以及部分2B、3B编码基因的片段P12X3C和包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C、3D以及部分2B、3B编码基因的片段P12X3C3D，将获得的基因片段直接/酶切后与同样处理的真核表达质粒连接，分别得到重组质粒pcDNA3.1/P12X3C和pcDNA3.1/P12X3C3D、pTARGET/P12X3C3D；对重组质粒进行序列测定、分析，并将重组质粒分别转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中FMDV抗原的表达，用电子显微镜观察病毒空衣壳的组装；为评价重组质粒作为DNA疫苗对实验动物及本动物的免疫效果，将重组质粒经肌肉注射方法接种豚鼠，并与酵母表达的纯化FMDV China99株3D蛋白及带有牛α干扰素的真核表达质粒pcDNA3.1/IFN分别/同时免疫，第二次免疫后第三周豚鼠攻以1OOID〓或1000ID〓的O型FMDV China99株；随后将质粒pcDNA3.1/P12X3C、pcDNA3.1/P12X3C3D与带有牛α干扰素的真核表达质粒pcDNA3.1/IFN同时免疫牛，三周后经牛舌皮攻以10〓ID〓的O型FMDV China99株。

By use of site mutation strategy and PCR technology, we obtained the gene P12X3C that includes full length PI, 2A, 3C and a part of 2B and 3B and the gene P12X3C3D that includes full length PI, 2A, 3C, 3D and a part of 2B and 3B. After being digested by restriction enzyme respectively, the gene P12X3C and the gene P12X3C3D were cloned into the pcDNA3.1 and pTARGET expression vector that were digested by the same enzyme. Recombinant plasmids were checked by restriction enzyme analysis and nucleic acid sequencing. Further more, recombinant plasmids were transfected into BHK-21 cells by using lipoid. The proteins of foot-and-mouth disease virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELlSA and fluoroscopy, and the capsid of FMDV was tested by electron microscope. In order to evaluate enhanced immune response of guinea pigs against FMDV, DNA vaccines which were designed to produce viral capsids lacking infectious viral nucleic acid and contained the gene P12X3C and the gene P 12X3C3D were injected respectively with FMDV 3D protein which was expressed in Pichia Pastoris Secreted expression System and purified or with pcDNA3.1/lFN which includes the gene IFN-a of cattle. Subsequently, Recombinant plasmids were injected to catties with or without pcDNA3.1/IFN. Anti-FMDV antibodies were detected by ELISA, and the T lymphocyte proliferation response was tested by MTT assay, neutralization antibodies liters were analyzed by micro-neutralization assay.

为研制带有O型口蹄疫病毒(Foot-and-Mouth Disease Virus，FMDV)China99株结构蛋白基因及多个非结构蛋白基因的DNA疫曲，本研究通过定点突变方法和PCR扩增方法，获得包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C以及部分2B、3B编码基因的片段P12X3C和包含有FMDV China99株结构蛋白P1、非结构蛋白2A、3C、3D以及部分2B、3B编码基因的片段P12X3C3D，将获得的基因片段直接／酶切后与同样处理的真核表达质粒连接，分别得到重组质粒pcDNA3.1／P12X3C和pcDNA3.1／P12X3C3D、pTARGET／P12X3C3D；对重组质粒进行序列测定、分析，并将重组质粒分别转染BHK-21细胞，通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中FMDV抗原的表达，用电子显微镜观察病毒空衣壳的组装；为评价重组质粒作为DNA疫苗对实验动物及本动物的免疫效果，将重组质粒经肌肉注射方法接种豚鼠，并与酵母表达的纯化FMDV China99株3D蛋白及带有牛α干扰素的真核表达质粒pcDNA3.1／IFN分别／同时免疫，第二次免疫后第三周豚鼠攻以100ID_(50)或1000ID_(50)的O型FMDV China99株：随后将质粒pcDNA3.1／P12X3C、pcDNA3.1／P12X3C3D与带有牛α干扰素的真核表达质粒pcDNA3.1／IFN同时免疫牛，三周后经牛舌皮攻以10~4ID_(50)的O型FMDV China99株。

The pHybLex/Zeo-Idl plasmid and the cDNA library plasmid were sequentially transformed into the yeast swains and screened to obtain Leu2^＋ and Leu2^＋LacZ^＋ clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.

方法PCR方法扩增Id1的编码序列并定向克隆入诱饵质粒pHybLex/Zeo，构建重组诱饵质粒pHybLex/Zeo-Id1，酶切鉴定后转化入酵母株EGY48/pSH18-34，检测重组诱饵质粒有无非特异激活报告基因Leu2、LacZ作用；扩增并提取成人肺组织文库质粒，将文库质粒及重组诱饵质粒转化入酵母细胞，依次筛选Leu2^＋，Leu2^＋LacZ^＋克隆；设置阴性及阳性对照，重复筛选Leu2＋LacZ＋克隆并排除假阳性克隆，获取真阳性文库质粒，进行测序和同源性比对。

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！