质粒

In order to get purified purified commercially luciferase, we selected whole gene fragment of Photinus pyralis as well as gene fragment deleting 18-nucleotide at 5'termination, which was synthesized according to P〓 template.

为了能得到商品纯的虫荧光素酶，将取自P〓质粒中的全长的北美萤火虫荧光素酶基因片断，及以P〓为模板合成的5＇端缺失18个核苷酸的虫荧光素酶基因片断。

MethodsThe D. bacteria - E. coli shuttle expression vector pZT17 was constructed based on plasmids of pUE30, pGBM5 and pKatCAT. Then pZT17 with lux+ from Photinus pyralis was used to transform into D. grandis and E.coli. The recombinant strains were induced separately.

以质粒pUE30、pGBM5及pKatCAT为基础，构建抗辐射菌属一大肠杆菌间的穿梭载体，将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体，并将该载体转化大肠杆菌诱导荧光素酶基因的表达。

Methods: Total RNA was extracted from esophageal carcinoma tissue, the primer containing special enzyme was designed, NY-ESO-1 fragment was amplified by RT-PCR, and then was cloned into the expression vector pET-15b by LP Recco PCR Cloning. The recombinants plasmid pET-15b-NY-ESO-1 were identified by restriction endonucleases digest analysis and sequence analysis.

从人新鲜的食管癌组织中提取总RNA，通过RT-PCR技术扩增NY-ES0-1基因3'端的340bp片段，采用靶向克隆法将目的基因插入到原核表达载体pET-15b中，得到重组表达质粒pET-15b-NY-ESO-1，经过筛选，挑选出阳性克隆进行XhoⅠ和BamHⅠ双酶切图谱分析、PCR检测和扩增产物核苷酸序列分析等，鉴定所构建的原核表达载体。

These patterns could result from REMI events, tandem integrations, or a combination of both.

这些现象可能是质粒多位点串联整合并且整合后发生重组的结果。

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:The gene of human ET1 was synthesized according to the preferential codons of E. coli, cloned into the EcoRI and SalI sites of vector pThioHisA. The recombinant plasmid pThioHisA-ET1 was constructed , sequenced and transformed into E.coli TOP10. Induced and expressed fusion protein were identified and analysed by 12% SDS-PAGE and densitometry analyses. After the elution, denature and renature, the fusion protein Thioredoxin-ET1 was obtained by ProBondTM chromatogragraphy. The purity of Thioredoxin-ET1 was detected by HPLC. Inoculate Thioredoxin-ET1 once per mouse every 2 weeks in 25, 50 and 100μg separately on 3 groups for 4 times. 10 days after last inoculation, we obtained venipuncture blood.

根据人ET1的多肽序列合成ET1基因，将其插入到pThioHisA的EcoRI和 SalI位点，重组质粒pThioHisA-ET1进行酶切鉴定及序列测定验证后转化TOP10，IPTG诱导的重组菌经SDS-PAGE检测融合蛋白Thioredoxin-ET1的表达量；表达的融合蛋白用ProBond亲合层析纯化并经HPLC鉴测其纯度；每只小鼠按25、50、100ug/次剂量的Thioredoxin-ET1每两周免疫一次，共4次，最后一次免疫10d后制备抗血清，经Western blot和ELISA检测证明Thioredoxin-ET1融合蛋白具有ET1免疫反应原性。

Cells were immediately fixed and processed for immuno?uorescence and tested for translocation ofβ-catenin.β-catenin and target genes of Wnt/β-catenin signaling COX-2, cyclinD1, c-fos, c-Jun mRNA expression were examined by RT-PCR. Saos-2 osteoblastic cells were co-transfected with a wild type or mutant Tcf luciferase reporter gene and Renilla luciferase plasmid. Posttransfection cells were subjected to 1 hour of 12% elongation.

作用1h、2h和4h后，用免疫荧光的方法检测β-catenin的表达和在细胞上定位的变化；用半定量RT-PCR检测β-catenin mRNA表达的变化；用质粒转染的方法检测荧光素酶报告基因Tcf转录的情况；用半定量RT-PCR检测Wnt/β-catenin通路下游基因COX-2，cyclinD1， c-fos， c-Jun mRNA表达的情况；用免疫共沉淀的方法检测β-catenin和E-Cadherin的结合情况。

After restimulation in vitro with HTHV plus rhuIL-2, the splenocytes either from HTHV infected mice or from pEF-BOS/HTHV-NP plasmid DNA immunized mice were used as effectors in 4 h 51 Cr release assay.

分别取HTHV感染小鼠的脾细胞和pEF-BOS/HTHV-NP质粒DNA免疫小鼠的脾细胞，经体外抗原和IL-2再刺激诱导产生CTL效应细胞，进行4h 51 Cr释放实验。

Through the test of retransformation with plasmid pUB110 fromtransformation with plasmid pUB110 from transferant and passage test,B.licheniformis 6104 and its mutant 610401 were proved to be a good receptorfor exogenous genes,especially for lysine synthetase expression.

通过质粒的再转化试验及传代稳定性试验，进一步证实B.licheniformis 6104及其突变菌株610401是较好的受体菌，尤其是用于赖氨酸合成酶基因的表达。

Rapid amplification of cDNA end:Retroverse transcription product of total RNA extracted from normal porcine tissue was used as the template,gene specific primers were designed and advantage 2 polymerase mix was used in PCR,of which using porcine genomic DNA as the template:forward primer was designed according to the acquired consensus region of human and pig FGL2 3′ sequences while reverse primer was designed from human FGL2 3′ end downstream sequence;TA cloning.

以猪正常小肠及心脏组织提取新鲜总RNA，反转录后作为模板，设计基因特异性引物，采用Advantage 2 聚合酶混合物进行PCR扩增；依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物，以人FGL2基因3′末端序列设计下游引物，以猪基因组DNA为模板采用Advantage 2 聚合酶混合物进行PCR反应；PCR载体重组质粒DNA亚克隆扩增。

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！