核染质

Three RNAi target sites (named as Abil1, Abil2, Abil3) targeting the E3B1 (NCBI: NM-024397), an identified target sites and positive control GAPDH-A were selected. The pGenesil-1 eukaryotic expression vectors with the neoR mark and GFP green fluorescent mark were selected to construct E3B1 RNAi plasmid. The effect of RNAi targeting various sites on E3B1 genes expression was evaluated by testing the transfection efficiency with fluorescence microscope and western blot. The most effective siRNA in inhibiting E3B1 genes were screened. The most effective siRNA in inhibiting E3B1 genes screened and used to transfect the neuronal cells. After that, the inhibitor of axon growth was added and the axon growth was observed. Result: The siRNA expressing plasmids targeting the E3B1 were constructed successfully.

选择针对E3B1(NCBI： NM-024397)RNAi的3个靶位点Abil1、Abil2、Abil3和1个不针对任何mRNA的RNAi靶位点以及甘油醛－3-磷酸脱氢酶，用带有neoR选择标志和GFP绿色荧光标志的真核表达载体pGenesil-1构建E3B1 RNAi质粒，分别转染培养的神经元，在荧光显微镜下观测转染率，经G418筛选得到单一的转染细胞，并用Western blot法检测各转染组神经元E3B1蛋白的表达情况，选出具有最佳抑制效应的siRNA转染神经元，加入轴突生长抑制物，观测轴突生长情况。

The pcDNA〓-apoE〓 plasmid was transfected to SK-N-SH Neuroblastoma cell by liposome. It could be screened by G〓. The immunohistochemical assay was shown that neuroblastoma cell transfected by pcDNA〓-apoE〓 expressed the recombinant apoE protein. The intracellular expressed recombinant protein could inhibit the death of neuroblastoma cell induced by beta amyloidal peptides 25-35 fragment.

用pcDNA〓—apoE〓真核表达载体与脂质体共转染神经成纤维胶质瘤细胞，用G〓选择压力筛选，细胞免疫荧光检测表明，pcDNA〓—apoE〓转染的神经成纤维胶质瘤细胞表达了apoE〓重组蛋白，这种内源性的apoE〓重组蛋白对25μM Aβ25—35多肽诱导的神经成纤维胶质瘤细胞的死亡具有拮抗作用。

The stable clones are further identified by RT-PCR and Western blot; 6 MTT assay is used to investigate the effect of ZNRD1 on the cell growth of cells (AGS, SGC7901, MKN28, NIH3T3, GES-1); 7 Soft agar assay is used to investigate the effect of ZNRD1 on the clonality of cells (AGS, MKN28); 8 Nude mice assay is used to investigate the effect of ZNRD1 on the cell growth of gastric cancer cells (AGS, MKN28); 9 Flow cytometry is used to investigate the effect of ZNRD1 on the cell cycle distribution of cells (AGS, MKN28, NIH3T3, GES-1); 10 Flow cytometry is used to investigate the effect of ZNRD1 on the cell apoptosis of cells (AGS, MKN28, NIH3T3); 11 MTT assay is used to investigate the effect of ZNRD1 on the drug sensitivity of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR) in vitro; 12 SRCA is used to investigate the effect of ZNRD1 on the drug sensitivity of gastric cancer cells (SGC7901, SGC7901/VCR) in vivo; 13 Flow cytometry is used to investigate the effect of ZNRD1 on adriamycin accumulation of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR); 14 Transmission electron microscope is used to investigate the effect of ZNRD1 on the sensitivity of SGC7901 cells towards drug-induced apoptosis; 15 Flow cytometry and DNA ladder assay are used to investigate the effect of ZNRD1 on the sensitivity of cells (SGC7901, SGC7901/VCR, HL-60/VCR) towards drug-induced apoptosis; 16 Microarray is used to investigate the profiling of ZNRD1-responsive genes in gastric cancer cells (AGS, MKN28, SGC7901, SGC7901/VCR); 17 RT-PCR and Western blot are used to identify the results of microarray; 18 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of cyclin D1; 19 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of MDR1; 20 Kinase assay is used to investigate the effect of ZNRD1 on the activity of cyclin E-CDK2 kinase; 21 The antisensenucleic acids of p21 is used to inhibit the expression of p21, and flow cytometry is used to investigate the effect of p21 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 22 The antisensenucleic acids of p27 is used to inhibit the expression of p27, and flow cytometry is used to investigate the effect of p27 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 23 Liposome is used to up-regulate the expression of Skp2, and flow cytometry is used to investigate the effect of Skp2 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 24 Western blot is used to investigate the effect of ZNRD1 on the stability of Skp2 and p27 in gastric cancer cells; 25 MVD assay is used to investigate the effect of ZNRD1 on the angiopoietic activity of gastric cancer cells; 26 ELISA is used to investigate the effect of ZNRD1 on the expression of VEGF165 in gastric cancer cells; 27 The roles of DARPP-32 in MDR of gastric cancer cells are investigated using gene transfection, MTT assay, SRCA, flow cytometry and DNA ladder assay.

应用杂交瘤技术制备ZNRD1的首个单克隆抗体；2）利用RT-PCR、Western blot和免疫组化检测ZNRD1在胃癌组织、胃炎组织、正常胃上皮组织、胃癌细胞和正常胃组织上皮细胞中的表达；3）构建ZNRD1的小干扰RNA载体，并测序鉴定；4）利用脂质体将ZNRD1的真核表达载体及其空载体转染胃癌细胞（AGS、SGC7901、MKN28）和小鼠成纤维细胞（NIH3T3），G418筛选后进行鉴定；5）利用脂质体将ZNRD1的小干扰RNA载体及其空载体转染药敏胃癌细胞（SGC7901）、正常胃组织上皮细胞（GES-1）、对长春新碱耐药的胃癌细胞（SGC7901/VCR）、药敏白血病细胞（HL-60）、对长春新碱耐药的白血病细胞（HL-60/VCR），G418筛选后进行鉴定；6）利用MTT实验检测ZNRD1高/低表达对细胞（AGS、SGC7901、MKN28、NIH3T3、GES-1）生长的影响；7）通过软琼脂克隆形成实验检测上调ZNRD1对AGS、MKN28细胞克隆形成能力的影响；8）通过裸鼠成瘤实验检测上调ZNRD1对AGS、MKN28细胞体内成瘤性的影响；9）通过流式细胞仪分析ZNRD1高/低表达对细胞（AGS、MKN28、NIH3T3、GES-1）的细胞周期的影响；10）通过流式细胞仪分析上调ZNRD1对细胞（AGS、MKN28、NIH3T3）的凋亡的影响；11）通过MTT实验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）体外药物敏感性的影响；12）通过肾包膜下移植法检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR）体内药物敏感性的影响；13）通过流式细胞仪分析ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）内阿霉素蓄积和泵出的影响；14）通过透射电镜检测上调ZNRD1对SGC7901细胞凋亡敏感性的影响；15）通过流式细胞仪和DNA梯度试验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60）凋亡敏感性的影响；16）通过基因芯片检测ZNRD1高/低表达对胃癌细胞内基因表达谱的影响；17）利用RT-PCR、Western blot对基因芯片的结果进行鉴定；18）利用报告基因实验检测ZNRD1对cyclin D1的启动子活性的调节作用；19）利用报告基因实验检测ZNRD1高/低表达对MDR1的启动子活性的调节作用；20）利用激酶试验检测ZNRD1对cyclin E-CDK2 激酶活力的影响；21）利用反义核酸技术抑制p21的表达；通过流式细胞仪检测抑制p21对ZNRD1介导的细胞周期阻滞的影响；22）利用反义核酸技术抑制p27的表达；通过流式细胞仪检测抑制p27对ZNRD1介导的细胞周期阻滞的影响；23）利用脂质体转染法上调Skp2的表达；通过流式细胞仪检测上调Skp2对ZNRD1介导的细胞周期阻滞的影响；24）利用Western blot检测ZNRD1对p27和Skp2的蛋白稳定性的影响；25）利用微血管密度实验检测ZNRD1对AGS、MKN28细胞裸鼠移植瘤微血管形成的影响；26）利用ELISA检测ZNRD1对AGS、MKN28细胞培养上清和移植瘤匀浆中VEGF165含量的影响；27）利用脂质体转染法、MTT实验、肾包膜下移植法、流式细胞仪和DNA梯度试验检测新耐药相关分子DARPP-32对细胞（SGC7901、SGC7901/VCR、对阿霉素耐药的胃癌细胞SGC7901/ADR）多药耐药表型的影响；利用脂质体转染法和MTT实验检测下调ZNRD1对DARPP-32介导的胃癌多药耐药的调控作用。

1,After transfected the mutated mtDNA of colorectal carcinoma,the mtDNA D-loop region of the transfected cells displays new mutation points.2,The external source pieces of the mutated mtDNA can integrate to nuclear genome after transfection.3,There's no differences in apoptosis between combinations after transfected the mutation of mtDNA in NIH3T3 and LST cells.4,The mutated mtDNA may affect the action mechanism of occurrence and development in colorectal carcinoma through affecting its mtDNA mutation or integrating exogenetic mtDNA to its nuclear which may cause the abnormal expression of oncogene or anti-oncogene.

（1）转染突变的大肠癌细胞mtDNA后转染细胞的mtDNA均可发生多处的突变位点。（2）通过转染后突变的外源性的mtDNA可以整合到核基因组内。（3）突变的mtDNA转染LST细胞及NIH3T3细胞后，不影响转染细胞的凋亡改变。（4）mtDNA的突变可能通过影响体细胞mtDNA的突变和通过外源性mtDNA在核内的整合从而影响癌基因或抑癌基因的表达异常，从而参与肿瘤的发生发展。线粒体DNA；D―环区；突变；质粒；pcDNA3.1；转染

The PSCA_3 fragment was selected for its superior expression level in eukaryotic cells.Then the sig-PSCA_3-Fc-GPI genetic fragment was cloned into pVAX1-neo-IRES-GM/B7 vector to construct the final immunological inhanced DNA vaccine pVAX1-PSCA_3-FcGB. Immunofluorescence and flow cytometry were used to confirm the expression of PSCA_3 fragment by transfected into Cos7 cell.Finally,the anti-tumor effect of pVAX1-PSCA_3-FcGB was tested in murine prostate cancer model generated by RM-1 cell line.The animal was immunized with pVAX1-PSCA_3-FcGB DNA vaccine by intramuscular injection plus electroporation,pVAX1 and pVAX1-PSCA_1-FcGB plasmid were used as control.The inhibitory effect of tumor was investigated by observion of forming time,volume and inhibition ratio of tumor.Results:DNA sequencing conformed that the heterological PSCA fusion antigen fragment which was synchronized by overlapping-extending-PCR,was consistent to design.Enzyme digestion analysis showed that the 1 to 4 copies heterological PSCA fusion antigen fragments were constructed successfully.

方法(1)检索GenBank，选择包含人主要T细胞抗原表位序列的人PSCA基因片段，应用异种化抗原设计技术，保留人T细胞抗原表位，设计异种化PSCA融合抗原片段；(2)根据核酸序列按中心模板法设计引物，应用重叠延伸PCR技术拼接合成异种化PSCA融合抗原片段基因，以PCR、限制性酶切和DNA序列测定法进行鉴定：(3)利用DNA限制性内切酶BssHⅡ和MluⅠ酶切后粘端互补的特点，采用同尾酶法构建1—4拷贝异种化PSCA融合抗原片段(PNCA_1-PSCA_4)，并将上述片段分别插入真核表达载体pCI-neo-Fc-GPI中，转染293T细胞，借助免疫荧光+流式细胞术考察插入片段表达效率，最终选定PSCA_3片段进行下一步研究；(4)将sig-PSCA_3-Fc-GPI基因片段自pCI-PSCA_3-Fc-GPI质粒上切下，插入pVAX1-neo-IRES—GM/B7载体中，构建免疫增效DNA疫苗pVAX1-PSCA_3-FcGB，并应用转染Cos7细胞+免疫荧光/流式细胞术方法鉴定其在真核细胞中的表达情况；(5)给8周龄雄性C57BL/6小鼠皮下种植RM-1细胞，制备小鼠前列腺癌模型，并采用股四头肌肌肉注射+电脉冲法(Electroporation，EP)接种DNA疫苗质粒pVAX1-PSCA_3-FcGB，同时接种pVAX1空载体质粒和pVAX1-PSCA_1-FcGB质粒作为对照，通过观察计算免疫动物的成瘤时间、肿瘤体积和抑瘤率，来评价该DNA疫苗在小鼠体内的抑瘤效果。

The proliferation of SGC-7901 cell line was markedly inhibited by shRNA-GC-CⅢ（ P .05）. There was changes in cell morphology, such as cell volume reduction, karyopyknosis and chromatin conglomeration. FCM showed that GC-C inhibition caused the appearance of sub-G 0 /G 1 peak which increased from 5.47%（48h after transfection） to 5.63%（72h after transfection）.

转染GC-C shRNA真核表达载体后SGC-7901细胞的增殖能力受到抑制（ P 。05）；细胞形态发生改变，细胞体积缩小，核固缩，染色质浓缩聚集于核膜；流式细胞术检测显示G 1 峰前出现亚二倍体峰，转染48、72h后凋亡率分别为5.47%和5.63%（ P nbsp； 0.05）。

Comparison from the Germany isolate, phocine distemper virus type Ⅱ(PDV-2) and vaccine strain, the major difference is the long signal peptide domain while the mature protein exhibit high identity, and all of the 13 serine residues, four potential asparagine glycosylation sites and two hydrophobic regions supposed to affect the fusion function are completely identical. These data supports the view that F protein is more conservative than H protein in CDV.

三、犬瘟热病毒中国分离株囊膜糖蛋白基因免疫小鼠诱发的抗体应答经一系列步骤将CDV-YZ0101株的两囊膜糖蛋白基因定向导入真核表达载体pcDNA3.1中，DNA测序和酶切分析筛选阳性重组质粒克隆pcDNA-H和pcDNA-F，以重组质粒DNA和脂质体共转染COS-7细胞，用间接免疫荧光试验证实转染的COS-7细胞的胞浆中分别表达了CDV的H和F蛋白。

Methods Fourteen mutated vpr fragments were selected from patients with HIV. Both eukaryotic expression vector pcDNA3.1 and PCR products were purified, double-cut by Hind Ⅲ and BamH Ⅰ, and the cut products were legated and transformed into competent cells JM109. The 14 reconstructed plasmids were transfected into Hela cells. Cells with pcDNA vpr-wt, pcDNA vpr-Fs and pcDNA3.1 blank cells, and without pcDNA3.1 cell were established.

以14个带有HIV-1vpr基因片段的PcD-NA3.1真核表达载体构建重组质粒，将其转染Hela细胞，并设立保守株vpr基因转染细胞、突变株vpr-FS基因转染细胞、空载体转染细胞和未转染细胞作为对照，经逆转录多聚酶链式反应检测目的基因转染成功后，Pi染色，用流式细胞仪检测被转染细胞的细胞周期分布和细胞凋亡率。

Methods 14 mutanted vpr fragments selected from Chines patients with HIV. Both eukaryotic expression vector pcDNA3.1 and PCR products purified , double-cut by HindⅢ and BamH and the cut products legated and were into competent cells JM109. The 14 reconstructed plasmids electronically transfected into hela cells, and established cells with pcDNA vprwt 、pcDNA vpr-Fs and pcDNA3.1 blank cells, and without pcDNA3.1 cell.

将14个带有HIV-1 vpr基因片段的pcDNA3.1真核表达载体构建重组质粒，将其转染Hela细胞，并设立保守株vp r基因转染细胞、突变株 vpr-FS基因转染细胞、空载体转染细胞和未转染细胞作为对照，经RT-PCR检测目的基因转染成功后，经Pi染色用流式细胞仪检测被转染细胞的细胞周期分布和细胞凋亡率。

Methods pVAX1-GRA8 was purified and transfected into Africa green monkey kindey vero cell lines,expression of GRA8 in vero cell lines were detected by RT-PCR and immunoblot with rabbits sera infected with Toxoplasma gondii.

大量提取纯化重组真核表达质粒pVAX1-GRA8，瞬时转染培养的vero E-6细胞，RT-PCR和免疫印迹法检测GRA8在细胞中的表达。

It has been put forward that there exists single Ball point and double Ball points on the symmetrical connecting-rod curves of equilateral mechanisms.

从鲍尔点的形成原理出发，分析对称连杆曲线上鲍尔点的产生条件，提出等边机构的对称连杆曲线上有单鲍尔点和双鲍尔点。

The factory affiliated to the Group primarily manufactures multiple-purpose pincers, baking kits, knives, scissors, kitchenware, gardening tools and beauty care kits as well as other hardware tools, the annual production value of which reaches US$ 30 million dollars.

集团所属工厂主要生产多用钳、烤具、刀具、剪刀、厨具、花园工具、美容套等五金产品，年生产总值3000万美元，产品价廉物美、选料上乘、质量保证，深受国内外客户的青睐