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The Charon library DNA was then digested with restriction enzymes BamH Ⅰ and Hind Ⅲ, and DNA fragments of length of about 0.5-1. 5kb was subcloned into plasmid pUC 18 which was also digested by BamH Ⅰ and Hind Ⅲ and dephosphorylated by alkaline phosphatese before being ligated with Charon fragments.

抽提λCharon文库总DNA，用BamHⅠ和HindⅢ完全酶切电泳，用低熔点琼脂糖回收长约0.5-1.5kb的DNA片段；连接到pUC18上，pUC DNA先用BamHⅠ和HindⅢ酶切，后用碱性磷酸酯酶脱磷。

The recombinant plasmid was expressed in the mammalian cells and the fusion proteins exhibited high phytase activity.

重组的pEGFP-N3-aPPA2在COS7细胞中正常表达并检测出高的植酸酶活性。

By using liposome-mediated gene transfection technology, plasmid pCMVSV40T/PUR containing SV40Tag was transfected in primary embryonic precartilaginous stem cells, while non-transfected cells served as negative controls.

利用脂质体介导基因转染技术将含有SV40Tag的质粒pCMVSV40T/PUR转染原代胚胎前软骨干细胞，以未转染细胞作阴性对照。

AIM: To detect the stability of the recombinant adenovirus plasmid containing major outer membrane protein gene from Chlamydia psittaci in HEK293 cells, to evaluate whether cross reactivity exists or not between recombinant adenovirus and positive serum of Egg Drop Symdrome, and to evaluate whether SPF chicks vaccinated with the recombinant adenovirus were protected or not when challenged with virulent CpL strain.

目的： 检测重组腺病毒质粒在HEK293细胞中的稳定性；重组腺病毒是否与减蛋综合征阳性血清发生中和反应，以及用重组腺病毒免疫SPF小鸡是否产生保护。

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。

Methods: We constructed a recomb ined adenoviral vector carrying CD or LacZ. 293 packaging cell was trans fected by the newly-constructed plasmid and generated the infectious virus.

构建含CD或LacZ基因的重组腺病毒载体，在293细胞内重组包装后，体外感染小鼠结肠癌细胞株C26，并给予不同浓度的前药氟胞嘧啶(5-FC)进行肿瘤杀伤效果观察。

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。

To express chicken osteoprotegerin in Pichia pastoris and determine its inhibitive effects on mature osteoclastic bone resorptive function in vitro. The chOPG cDNA encoding for 382 amino acid without signal peptice was subcloned into Pichia pastoris expression vector pPICZα-A. The constructed plasmid was transformed into yeast X-33 by electroporation. The recombinant transformants were selected by Zeocin. Induced by the addition of methanol every 24 hours, the product analyzed by SDS-PAGE was sized about 43kDa and 53kDa at a yield of 200mg per litter of culture. The result of Western blotting indicated that the recombinant protein had specific antigenicity mainly owing to heterogeneous glycosylation.

为了在毕赤酵母中分泌表达鸡骨保护素（osteoprotegerin，OPG），采用DNA重组技术将chOPG成熟肽cDNA片段插入毕赤酵母表达载体pPICZα-A中，以电穿孔法转化酵母X-33，用Zeocin 平板筛选重组子，经甲醇诱导表达后， SDS-PAGE 和免疫印迹分析表达产物，由于糖基化不同，表达产物有两种，其相对分子量分别为43kDa和53kDa，表达量约为200mg/L，经Western印迹验证，有较好的抗原性。

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！